New inhibitors for the keap1-nrf2 protein-protein interaction

ABSTRACT

Described herein are compounds, compositions and methods useful for inhibiting Kelch-like ECH-associated protein 1 (KEAP1). The compounds, compositions and methods described herein are useful for treating diseases, disorders or conditions associated with KEAP1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 62/984,010, filed Mar. 2, 2020, the contentof which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under CA200913 awardedby National Institutes of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

The present disclosure relates generally to compounds, compositions andmethods useful for inhibiting Kelch-like ECH-associated protein 1(KEAP1). For example, compounds, compositions and methods useful forinhibiting Nuclear factor erythroid-derived 2-related factor 2(NRF2)-KEAP1 interaction.

BACKGROUND

Nuclear factor erythroid-derived 2-related factor 2 (NRF2) is a masterregulator of cellular resistance to oxidative stress and cellular repair(Yonchuk, J. G. et al., J Pharmacol. Exp. Ther. 363, 114-125 (2017)).Under unstressed conditions, NRF2 is sequestered by Kelch-likeECH-associated protein 1 (KEAP1), an E3 ubiquitin ligase substrateadaptor, and targeted for degradation (Pallesen, J. S., Tran, K. T. &Bach, A. J. Med. Chem. 61, 8088-8103 (2018)). However, upon oxidativestress, reactive oxidants dissociate NRF2 from KEAP1 and NRF2translocates to the nucleus to activate its transcriptional program ofapproximately 250 genes (Davies, T. G. et al., J Med. Chem. 59,3991-4006 (2016)). The NRF2-KEAP1 pathway is critical in protecting thecell under oxidative stress and inflammation and is implicated in anumber of diseases (Cuadrado, A. et al., Nat. Rev. Drug Discov. 18,295-317 (2019)). There are ten drugs targeting KEAP1 that are inclinical trials and nine more that are at the preclinical stage(Cuadrado, A. et al., Nat. Rev. Drug Discov. 18, 295-317 (2019)).However, challenges regarding target specificity, pharmacodynamicproperties, efficacy and safety remain. Accordingly, there remains aneed in the art for compounds, compositions and methods inhibiting KEAP1for use in treating KEAP1 related diseases or conditions. The presentdisclosure addresses some of these needs.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides compounds of Formula (I):

-   -   wherein:        -   R¹ is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl;        -   R² is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl;        -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂; and        -   each R^(A) independently is H, substituted or unsubstituted            alkyl, substituted or unsubstituted alkenyl, substituted or            unsubstituted aryl, substituted or unsubstituted heteroaryl,            substituted or unsubstituted heterocyclyl, or substituted or            unsubstituted cyclyl,    -   or a pharmaceutically acceptable salt, solvate, hydrate,        polymorph, co-crystal, tautomer, stereoisomer, isotopically        labeled derivative, or prodrug thereof.

In one aspect, the disclosure provides compounds of Formula (II):

-   -   wherein:        -   A is substituted or unsubstituted arylene, substituted or            unsubstituted biarylene, or substituted or unsubstituted            heteroarylene;        -   R⁴ is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl; and        -   R⁵ is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl;        -   or a pharmaceutically acceptable salt, solvate, hydrate,            polymorph, co-crystal, tautomer, stereoisomer, isotopically            labeled derivative, or prodrug thereof.

In one aspect, the disclosure provides compounds of Formula (III):

-   -   wherein:        -   each A is independently substituted or unsubstituted aryl,            or substituted or unsubstituted heteroaryl;        -   R⁶ is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl; and        -   R⁷ is substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, or substituted or unsubstituted            heterocyclyl;            or a pharmaceutically acceptable salt, solvate, hydrate,            polymorph, co-crystal, tautomer, stereoisomer, isotopically            labeled derivative, or prodrug thereof.

In another aspect, the disclosure provides a compound selected fromGroup A, where the Group A comprises the following compounds:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof

It is noted that any reference to a compound of the disclosure includesa pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

The compounds described herein can be formulated into compositions orformulations. Accordingly, in another aspect provided herein ispharmaceutical composition comprising a compound described herein and apharmaceutically acceptable carrier or excipient.

The compounds of the disclosure can inhibit KEAP1. Accordingly, anotheraspect of the disclosure provides a method of inhibiting KEAP1. Themethod comprising contacting KEAP1 with a compound of the disclosure.

Still another aspect of the disclosure provides a method of inhibitingKEAP1-Nrf2 interaction. The method comprising contacting KEAP1 with acompound of the disclosure.

Still yet another aspect of the disclosure provides a method ofactivating Nrf2 The method comprising contacting KEAP1 with a compoundof the disclosure.

Compounds, composition or methods of inhibiting KEAP1 can be useful fortreating, preventing, or ameliorating a disease, disorder or conditionassociated with dysfunction of KEAP1 Nrf2 axis in a subject.Accordingly, another aspect of the disclosure provides a method oftreating a disease, disorder or condition associated with dysfunction ofthe KEAP1-Nrf2 axis. The method comprising administering to a subject inneed thereof a therapeutically effective amount of a compound of thedisclosure.

By a disease, disorder or condition associated with dysfunction of theKEAP1-Nrf2 axis is meant a disease or disorder whose pathology involvesa KEAP1-Nrf2 interaction. Accordingly, yet another aspect of thedisclosure provides a method of treating a disease, disorder orcondition associated with KEAP1-Nrf2 interaction. The method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of the disclosure.

In some embodiments, the disease, disorder or condition that can betreated with a compound, composition or method of the disclosure can beselected from the group consisting abdominal aortic aneurysm, acutekidney injury, adult brain glioblastoma, advanced solid tumors lymphoidmalignancies, aging, alcohol sensitivity, allergic, Alport syndrome,Alzheimer's disease, asthma, atopic asthmatics, autism spectrumdisorder, autosomal dominant polycystic kidney, Barrett esophagus,low-grade dysplasia, brain ischemia, breast cancer or breast neoplasm,cardiovascular risk, cataract surgery, cholelithiasis, cholestasis,chronic hepatitis c, chronic kidney disease, chronic lymphocyticleukemia, chronic renal insufficiency, chronic schizophrenia, chronicsubclinical inflammation, CKD associated with type 1 diabetes,cognition, colon cancer, COPD, corneal endothelial cell loss, crohn'sdisease, cutaneous t cell lymphoma, diabetes mellitus, diabeticnephropathy, diarrhea, endometriosis, environmental carcinogenesis,focal segmental glomerulosclerosis, Friedreich's ataxia, healthy,Helicobacter pylori infection, hepatic impairment, healthy, huntingtondisease, IgA nephropathy, inflammation and pain following ocularsurgery, insulin resistance, liver disease, lung cancer, majordepression, melanoma, metabolic syndrome x, mild cognitive impairment,mitochondrial myopathy, multiple sclerosis, neoplasms, nonalcoholicfatty liver or nonalcoholic steatohepatitis, noninsulin-dependent,nonischemic cardiomyopathy, obstructive sleep apnea, ocularinflammation, ocular pain, polymorphism, prediabetes, primary biliarycirrhosis, primary focal segmental glomerulosclerosis (FSGS), prostatecancer, psoriasis, psychosis, pulmonary arterial hypertension (pah),pulmonary hypertension, redox status, rheumatoid arthritis, rhinitis,schistosomiasis, schizophrenia, small lymphocytic lymphoma, subarachnoidhaemorrhage, and type 2 (type 2 diabetes).

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1H show docking poses (FIGS. 1A and 1B) and experimentalverification (FIGS. 1C-1H) of two exemplary compounds (iKeap1 andiKeap2). The docking poses (FIGS. 1A and 1Bb) were obtained from stage-2of the virtual screening. SPR steady-state binding curves are shown foriKeap1 (FIG. 1C) and iKeap2 (FIG. 1D), showing clear binding withnanomolar K_(d). Shown is one representative data from three independentexperiments (n=3) with similar results. Ligand-detected NMR experiments,CPMG-R2 and STD-NMR (FIGS. 1E and 1F) confirm the binding of the twocompounds. The two hits were also functional in the FP assay (FIGS. 1Gand 1H) confirming that the compounds displace the peptide. The FP datashown here is from three technical replicates and the curve was fittedto the average value of the three the technical replicates. The mean andthe standard deviation for the individual data points are shown. The FPwas repeated independently twice with similar results and onerepresentative result is shown here.

FIGS. 2A and 2B show binding of the NRF2 peptide to KEAP1 as assayed byFP (FIG. 2A) and BLI (FIG. 2B). In FIG. 2A, for the FP assay aTAMRA-tagged NRF2 peptide and for the BLI assay a Biotin-tagged NRF2peptide were used. The FP assay was performed with three technicalreplicates per point. The mean and standard deviation are shown for eachtitration point, along with the fitted curve. Two independentexperiments (n=2) were performed, each with similar results and onerepresentative result is shown here. In FIG. 2B, for the BLI assay abiotin-tagged NRF2 peptide was used. The BLI experiment was repeatedindependently twice (n=2) with similar results and one representativeresult is shown here.

FIGS. 3A-3D show comparison of exemplary compound (iKeap1) with apreviously identified displacer C17. FIG. 3A shows crystal structure(PDB ID: 5FNQ⁹) of KEAP1 with its ligand removed, the structure used forthe primary virtual screening procedure. FIG. 3B shows structure ofKEAP1 (PDB code 4IQK) with ligand C17 (Table 2), which is also shown inFIG. 3D. FIG. 3C shows iKeap1, the best binder as accessed by array ofexperimental validations, is similar to compound C17 previouslyidentified by experimental methods (FIG. 3D). Though iKeap1 and C17 looksimilar they differ in a number of aspects in their core scaffold(therefore analogues of the two compounds cover distinct chemicalspaces, assuming the analogues retain the core scaffold of the parentcompound). This similarity, as well as the fact that the predicteddocking positions (FIG. 1A) of both ligands (FIG. 3B) are nearlyidentical, is an additional evidence that iKeap1 is binding at thepredicted site.

FIGS. 4A-4H shows the difference between binders and displacers for twoexemplary compounds, iKeap8 and. SPR confirms that both iKeap8 andiKeap9 bind KEAP1 (FIGS. 4A and 4B) with similar K_(d) values. Shown arerepresentative results from the SPR assay for iKeap8 and iKeap9. Foreach compound, three independent SPR experiments were performed, eachwith similar results and one representative result is shown here.Ligand-detected NMR experiments shows that both iKeap8 and iKeap9 bindto KEAP1 (FIGS. 4C and 4D). However, FP (FIGS. 4E and 4F) and BLI (FIGS.4G and 4H) assays show that iKeap8 is able to displace the NRF2 peptidewhile iKeap9 is not able to effectively displace the NRF2 peptide. Thefluorescence polarization (FP) assay was performed with three technicalreplicates per concentration measured. The mean and standard deviationare shown for each titration point, along with the fitted curve.

FIGS. 5A-5F shows two more exemplary displacers, iKeap7 and iKeap22,both of which were confirmed as binders by SPR (top panels).Ligand-detected NMR experiments shows that both iKeap7 and iKeap22 bindto KEAP1 (FIGS. 4C and 4D). iKeap7 is confirmed to be a displacer of theNRF2 peptide by both FP (bottom left panel) and BLI (not shown). Sincethe FP experiments on iKeap22 were affected by autofluorescence, BLI(bottom right panel) was needed to confirm that this compounds is adisplacer. The FP assay was performed with three technical replicatesper concentration measured. The mean and standard deviation are shownfor each titration point, along with the fitted curve. Two independentBLI experiment were performed with similar results and onerepresentative result shown here.

FIG. 6 shows the docking pose of one of the hit compounds (iKeap9,ball-and-stick representation) bound to KEAP1, together with the NRF2peptide (PDB ID: 4IFL; peptide in violet). iKeap9 is a tight binder (180nM by steady-state SPR) but cannot displace NRF2. The left figure showsthe top view, while the right figure shows the side view of thecross-section of KEAP1 along the central plane. The violet box in rightfigure indicates the docking region (where the ligands were allowed tobind) which was used in the virtual screening. The site of interestincludes a part of the deep pocket/tunnel of the β-barrel-shaped KEAP1,since it can allow ligands to bind more tightly by insertion into thechannel than on a shallow surface. However, the deep tunnel is largelynon-overlapping with the peptide binding site (which binds to theentrance site of the tunnel). Thus, binding molecules might onlypartially interfere with the peptide binding, which might reduce oreliminate the ability of small molecule binders to displace the peptide.The ability of a small molecule to displace the peptide is hard topredict, and was not attempted in this study. In some cases, smallmolecules can also act as molecular glues and strengthen the interactionbetween NRF2 and KEAP1.

FIGS. 7A-7D are ¹H-¹³C HMQC experiments showing the binding of the NRF2peptide (FIGS. 7A and 7C), iKeap1 (FIG. 7B) and iKeap2 (FIG. 7D) toKEAP1 as monitored by chemical shift perturbation to the methylresonances of Ile, Leu and Val of KEAP1. Upon addition of iKeap1 andiKeap2 we see selective and specific changes to a subset of resonancesand these correlate to the changes we observe when we add the NRF2peptide. The rest of the resonances are largely unaffected. Theindicates that the protein is folded and does not aggregate after theaddition of the compounds.

FIGS. 8A-8D are ¹H-¹³C HMQC experiments showing the binding of the NRF2peptide (FIGS. 8A and 8C), iKeap8 (FIG. 8B) and iKeap9 (FIG. 8D) toKEAP1 as monitored by chemical shift perturbation to the methylresonances of Ile, Leu and Val of KEAP1. Upon addition of iKeap8 andiKeap9 we see selective and specific changes to a subset of resonancesand these correlate to the changes we observe when we add the NRF2peptide. The rest of the resonances are largely unaffected. Theindicates that the protein is folded and does not aggregate after theaddition of the compounds.

FIGS. 9A-9D are ¹H-¹³C HMQC experiments showing the binding of the NRF2peptide (FIGS. 9A and 9C), iKeap7 (FIG. 9B) and iKeap22 (FIG. 9D) toKEAP1 as monitored by chemical shift perturbation to the methylresonances of Ile, Leu and Val of KEAP1. Upon addition of iKeap7 andiKeap22 we see selective and specific changes to a subset of resonancesand these correlate to the changes we observe when we add the NRF2peptide. The rest of the resonances are largely unaffected. Theindicates that the protein is folded and does not aggregate after theaddition of the compounds.

FIG. 10 shows NMR solubility assay of iKeap1. Determination of thesolubility of iKeap1 via an NMR solubility assay as described in[LCG+13]. As can be seen in the figure, the NMR intensity of iKeap1remains linear over the range of concentrations measured here,indicating that iKeap1 does not aggregate at these concentrations.

FIG. 11 shows NQO1 assay results.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

Various aspects described herein are based on inventors' discovery interalia of compounds that inhibit KEAP1. For example, the inventors havediscovered compounds that can disrupt or inhibit KEAP1-Nrf2interactions.

Accordingly, in one aspect provided herein is a compound of Formula (I).

In some embodiments of any one of the aspects, R¹ in compounds ofFormula (I) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, R¹ is phenyl, quinolinyl,quinolonyl or chromenyl, each of which can be optionally substituted. Insome embodiments, R¹ is phenyl or phenyl substituted with an alkoxygroup. In some embodiments, R¹ is phenyl,4-[2-(2,3-Dihydro-1-benzofuran-5-yl)ethoxy]-phenyl, 3-benzyloxy-phenyl,quinolinyl, quinolonyl, 7-methyl-2-oxo-1,2-dihydro-3-quinolinyl,4H-chromenyl, or 7-hydroxy-4-oxo-4H-chromenyl. Preferably, R¹ is4-[2-(2,3-Dihydro-1-benzofuran-5-yl)ethoxy]-phenyl, 3-benzyloxy-phenyl,7-methyl-2-oxo-1,2-dihydro-3-quinolinyl, or7-hydroxy-4-oxo-4H-chromenyl.

In some embodiments of any one of the aspects, R² in compounds ofFormula (I) is phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl,imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl,tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl,or indanyl. For example, R¹ is phenyl, pyridinyl, pyrimidinyl, furanyl,naphthyl or anthracenyl, each of which can be optionally substituted. Insome embodiments, R² is phenyl, which can be optionally substituted. Insome embodiments, R² is 4-hydroxyphenyl, which can be optionally furthersubstituted. In some preferred embodiments, R² is 4-hydroxyphenyl or3,4-dihydroxyphenyl.

In some embodiments of any one of the aspects, R³ in compounds ofFormula (I) is H or OR^(A), where R^(A) is H or C₁-C₆alkyl. For example,R³ is H, OH, methoxy, ethoxy, propoxy, isopropoxy or butoxy. In somepreferred embodiments, R³ in compounds of Formula (I) is H or OH.

In some embodiments of any one of the aspects, R^(A) in compounds ofFormula (I) is H, substituted or unsubstituted alkyl. For example, R^(A)is H or C₁-C₆alkyl. In some embodiments, R^(A) is H, methyl, ethyl,propyl, isopropyl, butyl or pentyl. Preferably R^(A) is H.

Exemplary compounds of Formula (I) include, but are not limited to, thefollowing:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

In one aspect, provided herein are compounds of Formula (II).

In some embodiments, of any one of the aspects, A in compounds ofFormula (II) is benzene, pyridine, pyrimidine, furan, naphthalene,quinolone, quinolone, or anthracene, each of which can be optionallysubstituted. For example, A can be benzene, pyridine, naphthalene oranthracene, each of which can be optionally substituted. In someembodiments, A is benzene, methylbenzene, methoxybenzene,aminodimethylbenzene, naphthalene, or3-sulfonyl-1-hydroxy-6-aminonaphthalene.

In some embodiments, of any one of the aspects, R⁴ in compounds ofFormula (II) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, R⁴ is phenyl, naphthyl, quinolinyl,quinolonyl or chromenyl, each of which can be optionally substituted. Insome embodiments, R⁴ is phenyl or naphthyl, each of which can beoptionally substituted. For example, R⁴ is phenyl, 4-Sulfonylphenyl,4-sulfonyl-2-methylphenyl, 2-methylphenyl, 3-carboxy,5-sulfonyl-4-hydroxyphenyl, phenylazophenyl, (4-methylphenyl)azophenyl,3-(4-methylphenyl)azophenayl,(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,5-(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,phenylazo-2-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azophenyl, nitrophenyl, 4-nitophenyl,(3-carboxy, 5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, phenylazonaphthyl,nitrophenylazonaphthyl, (4-nitrophenyl)azonaphthyl,5-(nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,5-(4-nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,hydroxysulfonylnaphthyl, aminosulfonyl, aminohydroxysulfonylnaphthyl,1-sulfonyl-4-hydroxynaphthyl, 3-sulfonyl-1-hydroxynaphthyl,1-hydroxysulfonylnaphthyl, 1-hydroxy-3-sulfonylnaphthyl,1-hydroxy-5-sulfonylnaphthyl, 1-hydroxy-3-sulfonyl-7-aminonaphthyl,1-aminosulfonylnaphthyl, 1-amino-3-sulfonylnaphthyl,1-amino-7-sulfonylnaphthyl, 4-amino-6-sulfonylnaphthyl,(methylphenyl)aminosulfonylnaphthyl,4-(methylphenyl)aminosulfonylnaphthyl,(methylphenyl)amino-5-sulfonylnaphthyl,4-(methylphenyl)amino-5-sulfonylnaphthyl,1-(methylphenyl)amino-8-sulfonylnaphthyl,(4-methylphenyl)aminosulfonylnaphthyl,4-(4-methylphenyl)aminosulfonylnaphthyl,(4-methylphenyl)amino-5-sulfonylnaphthyl,4-(4-methylphenyl)amino-5-sulfonylnaphthyl, or1-(4-methylphenyl)amino-8-sulfonylnaphthyl, each of which can beoptionally further substituted.

In some embodiments, of any one of the aspects, R⁵ in compounds ofFormula (II) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, R⁵ is phenyl, naphthyl, quinolinyl,quinolonyl or chromenyl, each of which can be optionally substituted. Insome embodiments, R⁵ is phenyl or naphthyl, each of which can beoptionally substituted. For example, R⁵ is phenyl, 4-Sulfonylphenyl,4-sulfonyl-2-methylphenyl, 2-methylphenyl, 3-carboxy,5-sulfonyl-4-hydroxyphenyl, phenylazophenyl, (4-methylphenyl)azophenyl,3-(4-methylphenyl)azophenayl,(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,5-(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,phenylazo-2-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azophenyl, nitrophenyl, 4-nitophenyl,(3-carboxy, 5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, phenylazonaphthyl,nitrophenylazonaphthyl, (4-nitrophenyl)azonaphthyl,5-(nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,5-(4-nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,hydroxysulfonylnaphthyl, aminosulfonyl, aminohydroxysulfonylnaphthyl,1-sulfonyl-4-hydroxynaphthyl, 3-sulfonyl-1-hydroxynaphthyl,1-hydroxysulfonylnaphthyl, 1-hydroxy-3-sulfonylnaphthyl,1-hydroxy-5-sulfonylnaphthyl, 1-hydroxy-3-sulfonyl-7-aminonaphthyl,1-aminosulfonylnaphthyl, 1-amino-3-sulfonylnaphthyl,1-amino-7-sulfonylnaphthyl, 4-amino-6-sulfonylnaphthyl,(methylphenyl)aminosulfonylnaphthyl,4-(methylphenyl)aminosulfonylnaphthyl,(methylphenyl)amino-5-sulfonylnaphthyl,4-(methylphenyl)amino-5-sulfonylnaphthyl,1-(methylphenyl)amino-8-sulfonylnaphthyl,(4-methylphenyl)aminosulfonylnaphthyl,4-(4-methylphenyl)aminosulfonylnaphthyl,(4-methylphenyl)amino-5-sulfonylnaphthyl,4-(4-methylphenyl)amino-5-sulfonylnaphthyl, or1-(4-methylphenyl)amino-8-sulfonylnaphthyl, each of which can beoptionally further substituted.

In compounds of Formula (II), R⁴ and R⁵ can be same or different. Insome embodiments, R⁴ and R⁵ are same. In some other embodiments, R⁴ andR⁵ are different.

In compounds of Formula (II), R⁴ and R⁵ can be same or different. Insome embodiments, R⁴ and R⁵ are same. In some other embodiments, R⁴ andR⁵ are different.

Exemplary compounds of Formula (II) include, but are not limited to, thefollowing:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

In one aspect, provided herein are compounds of Formula (III).

In some embodiments, of any one of the aspects, each A in compounds ofFormula (III) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, each A can be phenyl, which can beoptionally substituted. In some embodiments, each is independentlyphenyl, sulfonylphenyl, or 2-sulfonylphenyl, each of which can beoptionally further substituted.

In some embodiments, of any one of the aspects, R⁶ in compounds ofFormula (II) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, R⁶ is phenyl, naphthyl, quinolinyl,quinolonyl or chromenyl, each of which can be optionally substituted. Insome embodiments, R⁶ is phenyl or naphthyl, each of which can beoptionally substituted. For example, R⁶ is aminonaphthyl,1-aminonaphthyl, 4-aminonaphthyl, 1-hydroxynaphthyl, 4-hydroxynaphthyl,phenyl, 2-hydroxy-1-carboxyphenyl, 3-hydroxy-4-carboxyphenyl,4-Sulfonylphenyl, 4-sulfonyl-2-methylphenyl, 2-methylphenyl, 3-carboxy,5-sulfonyl-4-hydroxyphenyl, phenylazophenyl, (4-methylphenyl)azophenyl,3-(4-methylphenyl)azophenayl,(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,5-(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,phenylazo-2-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azophenyl, nitrophenyl, 4-nitophenyl,(3-carboxy, 5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, phenylazonaphthyl,nitrophenylazonaphthyl, (4-nitrophenyl)azonaphthyl,5-(nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,5-(4-nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,hydroxysulfonylnaphthyl, aminosulfonyl, aminohydroxysulfonylnaphthyl,1-sulfonyl-4-hydroxynaphthyl, 3-sulfonyl-1-hydroxynaphthyl,1-hydroxysulfonylnaphthyl, 1-hydroxy-3-sulfonylnaphthyl,1-hydroxy-5-sulfonylnaphthyl, 1-hydroxy-3-sulfonyl-7-aminonaphthyl,1-aminosulfonylnaphthyl, 1-amino-3-sulfonylnaphthyl,1-amino-7-sulfonylnaphthyl, 4-amino-6-sulfonylnaphthyl,(methylphenyl)aminosulfonylnaphthyl,4-(methylphenyl)aminosulfonylnaphthyl,(methylphenyl)amino-5-sulfonylnaphthyl,4-(methylphenyl)amino-5-sulfonylnaphthyl,1-(methylphenyl)amino-8-sulfonylnaphthyl,(4-methylphenyl)aminosulfonylnaphthyl,4-(4-methylphenyl)aminosulfonylnaphthyl,(4-methylphenyl)amino-5-sulfonylnaphthyl,4-(4-methylphenyl)amino-5-sulfonylnaphthyl, or1-(4-methylphenyl)amino-8-sulfonylnaphthyl, each of which can beoptionally further substituted. Preferably, R⁶ is aminonaphthyl,1-aminonaphthyl, 4-aminonaphthyl, 1-hydroxynaphthyl, 4-hydroxynaphthyl,phenyl, 2-hydroxy-1-carboxyphenyl, or 3-hydroxy-4-carboxyphenyl.

In some embodiments, of any one of the aspects, R⁷ in compounds ofFormula (III) is phenyl, pyridinyl, pyrimidinyl, furanyl, quinolinyl,quinolonyl, naphthyl, anthracenyl or chromenyl, each of which can beoptionally substituted. For example, R⁷ is phenyl, naphthyl, quinolinyl,quinolonyl or chromenyl, each of which can be optionally substituted. Insome embodiments, R⁷ is phenyl or naphthyl, each of which can beoptionally substituted. For example, R⁷ is aminonaphthyl,1-aminonaphthyl, 4-aminonaphthyl, 1-hydroxynaphthyl, 4-hydroxynaphthyl,phenyl, 2-hydroxy-1-carboxyphenyl, 3-hydroxy-4-carboxyphenyl,4-Sulfonylphenyl, 4-sulfonyl-2-methylphenyl, 2-methylphenyl, 3-carboxy,5-sulfonyl-4-hydroxyphenyl, phenylazophenyl, (4-methylphenyl)azophenyl,3-(4-methylphenyl)azophenayl,(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,5-(4-methylphenyl)azo-2-amino-3,6-dimethylphenyl,phenylazo-2-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azophenyl, nitrophenyl, 4-nitophenyl,(3-carboxy, 5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-methylphenyl, (3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, 4-(3-carboxy,5-sulfonyl-4-hydroxyphenyl)azo-3-methylphenyl, phenylazonaphthyl,nitrophenylazonaphthyl, (4-nitrophenyl)azonaphthyl,5-(nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,5-(4-nitrophenyl)azo-3-sulfonyl-1-hydroxy-6-aminonaphthyl,hydroxysulfonylnaphthyl, aminosulfonyl, aminohydroxysulfonylnaphthyl,1-sulfonyl-4-hydroxynaphthyl, 3-sulfonyl-1-hydroxynaphthyl,1-hydroxysulfonylnaphthyl, 1-hydroxy-3-sulfonylnaphthyl,1-hydroxy-5-sulfonylnaphthyl, 1-hydroxy-3-sulfonyl-7-aminonaphthyl,1-aminosulfonylnaphthyl, 1-amino-3-sulfonylnaphthyl,1-amino-7-sulfonylnaphthyl, 4-amino-6-sulfonylnaphthyl,(methylphenyl)aminosulfonylnaphthyl,4-(methylphenyl)aminosulfonylnaphthyl,(methylphenyl)amino-5-sulfonylnaphthyl,4-(methylphenyl)amino-5-sulfonylnaphthyl,1-(methylphenyl)amino-8-sulfonylnaphthyl,(4-methylphenyl)aminosulfonylnaphthyl,4-(4-methylphenyl)aminosulfonylnaphthyl,(4-methylphenyl)amino-5-sulfonylnaphthyl,4-(4-methylphenyl)amino-5-sulfonylnaphthyl, or1-(4-methylphenyl)amino-8-sulfonylnaphthyl, each of which can beoptionally further substituted. Preferably, R⁷ is aminonaphthyl,1-aminonaphthyl, 4-aminonaphthyl, 1-hydroxynaphthyl, 4-hydroxynaphthyl,phenyl, 2-hydroxy-1-carboxyphenyl, or 3-hydroxy-4-carboxyphenyl.

In compounds of Formula (III), R⁶ and R⁶ can be same or different. Insome embodiments, R⁶ and R⁷ are same. In some other embodiments, R⁶ andR⁷ are different

Exemplary compounds of Formula (III) include, but are not limited to,the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

In Table 1, some exemplary compounds of the disclosure are described bytheir Simplified Molecular Input Line Entry System (SMILES). SMILESallows rigorous structure specification by use a compact use of naturalgrammar as described in detail by D. Weiniger “SMILES, a ChemicalLanguage and Information System. 1. Introduction to Methodology andEncoding Rules” J. Chem. Inf. Comput. Sci., Vol. 28, No. 1, 1988, pages31-36; the entirety of which are incorporated herein by reference

TABLE 1 Some exemplary compounds of the disclosure Compound SMILESiKeap28 CC1═CC═C(C(═O)NC(═CC2═CC═C(C3═CC═CC([N+](═O)[O−])═C3)O2)C(═O)NCC2CCCO2)C═C1 iKeap2O═C(O)c1ccc(NC2═C/C(═N\S(═O)(═O)c3ccc4ccccc4c3)c3ccccc3C2═O)cc1 iKeap24O═C(O)C(SC1═NC(═O)C2(NN1)c1ccccc1-c1ccccc12)SC1═NC(═O)C2(NN1)c1ccccc1-c1ccccc12 iKeap9O═S(═O)(N═C1CCCCCN1)C1═CC═C2SC(NS(═O)(═O) C3═CC═C4C═CC═CC4═C3)═NC2═C1iKeap20 Cc1cc2oc(═O)cc(COC(═O)[C@H]3CCCN(C(═O)c4ccc5[nH]ncc5c4)C3)c2cc1C(C)C iKeap4 O═C1OC(c2ccc([N+](═O)[O−])cc2)═N/C1═C\c1cccc2ccccc12iKeap29 COc1ccc(NS(═O)(═O)c2ccc(/N═C\c3c4ccccc4nc4ccccc43)cc2)nn1iKeap27 Cc1ccc(C)c(N2C(═O)c3ccc(-c4nc(-c5ccc6[nH]nnc6c5)no4)cc3C2═O)c1iKeap75 Cc1cccn2c(═O)c3c(nc12)N1CCCCC[C@H]1[C@]1(C3)C(═O)N(Cc2ccccc2Cl)C(═O)N═C1O iKeap51O═C(Cn1nc(-c2ccccc2)ccc1═O)Nc1cccc(Oc2ccc([N+](═O)[O−])cc2)c1 iKeap12O═C(c1ccccc1)C1═C[C@@H]2[C@@H]3C(═O)N(c4cccc5ccccc54)C(═O)[C@@H]3[C@@H](C(═O)3ccc(Cl)cc3)N2C═C1 iKeap18O═S(═O)(O)c1ccc(/N═N\c2ccc(/N═N\c3ccc(O)c4cccc(S(═O)(═O)O)c34) cc2)cc1iKeap26 O═C1C[C@H](C(═O)N2CCC[C@H](NC(═O)c3cc4ccccc4o3)C2)c2ccc(F) cc2N1iKeap36 O═C(NN═CC1═C(O)C([N+](═O)[O−])═CC(Cl)═C1)C1═CC(C2═CC═CC═N2)═NC2═CC═CC═C12 iKeap52Cc1ccc2cc([C@H]3CC(═O)Oc4cc(O)c5c(═O)c(O)c(-c6ccc(O)c(O)c6)oc5c43)c(═O)[nH]c2c1 iKeap7CC1═NN(c2ccccc2)C(═O)/C1═C\c1ccc(-c2cc(C(═O)O)ccc2Cl)o1 iKeap31O═C1C[C@H](c2ccc(OCCc3ccc4c(c3)CCO4)cc2)c2c(cc(O)c3c(═O)c(O)c(-c4ccc(O)c(O)c4)oc32)O1 iKeap16O═C(c1ccccc1)c1cc(-c2cc(═O)cc(-c3cc(C(═O)c4ccccc4)c(O)cc3O) o2)c(O)cc1OiKeap34 CC(C)c1ccc(COc2cccc([C@H]3CC(═O)Oc4cc(O)c5c(═O)c(O)c(-c6ccc(O)c(O)c6)oc5c43)c2)cc1 iKeap13O═C1OC(c2cccc3ccccc32)═N/C1═C\c1ccc(-c2cccc(C(F)(F)F)c2)o1 iKeap62COc1ccc2occ([C@@H]3CC(═O)Oc4cc(O)c5c(═O)cc(-c6ccc(O)c(O)c6)oc5c43)c(═O)c2c1 iKeap69CC1═CC═C(S(═O)(═O)NC2═CC(═NS(═O)(═O)C3═C(C)C═C(C)C═C3C)C3═CC═CC═C3C2═O)C═C1 iKeap39Cc1cccn2c(═O)c3c(nc12)N1CCCCC[C@H]1[C@]1(C3)C(═O)N (Cc2ccccc2)C(═O)N═C1OiKeap68 CC1═NOC(NS(═O)(═O)C2═CC═C(NC(═O)CC3═COC4═CC═C(C(C)C)C═C34)C═C2)═C1C iKeap40Cc1ccc(N2C(═O)[C@@H]3N═NN(CC(═O)N4N═C5/C(═C/c6ccccc6)CCC[C@@H]5[C@H]4c4ccccc4)[C@@H]3C2═O)cc1 iKeap5Nc1ccc2cc(S(═O)(═O)O)c(/N═N/c3ccc(/N═N\c4ccc(N)c5cc(S(═O)(═O)O)ccc45)c4ccccc34)c(O)c2c1 iKeap30Cc1ccc(/N═N\c2cc(C)c(N)c(/N═N\c3ccc(/N═N\c4cc(C(═O)O)c(O)c(S(═O)(═O)O)c4)c(C)c3)c2N)cc1 iKeap56COc1ccc(C(═O)C2═C(O)C(═O)N(c3nnc(SCc4cccc5ccccc54)s3)[C@H]2c2cccc(Oc3ccccc3)c2)cc1OC iKeap35CC1═NN(C2═CC═CC═C2)C(NC2═CC═C3C(═N2)OC2═NC(NC4═CC(C)═NN4C4═CC═CC═C4)═CC═C2C3C2═CC═C(Cl) C([N+](═O)[O−])═C2)═C1iKeap57 Cc1ccc(/C═C2\CCC[C@@H]3C2═NN(C(═O)CN2N═N[C@@H]4C(═O)N(c5cccc(F)c5)C(═O)[C@H]42)C@@H]3c2ccc(C)cc2)cc1 iKeap6Cc1ccc(Nc2ccc(/N═N\c3ccc(/N═N\c4cccc(S(═O)(═O)O)c4)c4ccccc34)c3cccc(S(═O)(═O)O)c23)cc1 iKeap11COc1cc(/N═N\c2ccc(S(═O)(═O)O)cc2)ccc1/N═N\c1ccc2c(cccc2S(═O)(═O) O)c1OiKeap77 O═C1C[C@@H](c2ccccc2)CC2═C1[C@@H](c1cccc(Oc3ccccc3)c1)Nc1ccccc1N2 iKeap38O═S(═O)(O)c1cc(/N═N/c2c(O)ccc3ccccc32)ccc1/C═C\c1ccc(/N═N\c2c(O)ccc3ccccc32)cc1S(═O)(═O)O iKeap23O═C(O)c1cc(/N═N\c2ccc(/C═C\c3ccc(/N═N\c4ccc(O)c(C(═O)O)c4)cc3S(═O)(═O)O)c(S(═O)(═O)O)c2)ccc1O iKeap54O═C(N/N═C/c1cc([N+](═O)[O−])cc([N+](═O)[O−])c1O)c1cc(-c2ccccc2)nc2ccccc21 iKeap14 O═[N+]([O−])c1ccc(Nc2ccc(Oc3ccc(Nc4ccc([N+](═O)[O−])c5nonc54)cc3)cc2)c2nonc21 iKeap1Cc1ccc(S(═O)(═O)Oc2nc3nc4ccccc4nc3nc2OS(═O)(═O)c2ccc(C)cc2)cc1 iKeap46Cc1nnnn1-c1cccc(NC(═O)c2c3c(nc4ccccc24)/C(═C\c2ccco2)CC3)c1 iKeap60O═C1N[C@@H](Cc2nc(-c3cccc(Cn4cnc5ccccc54)c3)no2)C(═O)Nc2ccccc21 iKeap50Cc1ccnc(NS(═O)(═O)c2ccc(/N═C\c3c4ccccc4nc4cc(Cl)ccc34)cc2)n1 iKeap32O═C(Cc1ccc(Cl)cc1)Nc1cccc(-c2ccc3nnc(-c4cccnc4)n3n2)c1 iKeap67O═C([C@H]1C[C@H]2CCCC[C@@H]2N1c1ncccn1)N1CC═C (c2c[nH]c3cc(F)ccc23)CC1iKeap55 Nc1ccc2c(O)c(/N═N\c3ccc(/N═N\c4ccc(S(═O)(═O)O)cc4)cc3)c(S(═O)(═O)O)cc2c1/N═N\c1ccc([N+](═O)[O−])cc1 iKeap3Cc1cc(/N═N\c2ccc(S(═O)(═O)O)cc2C)ccc1/N═N\c1cc(S(═O)(═O)O) c2ccccc2c1OiKeap25 O═S(═O)(O)c1cc(O)c2c(c1)cc(S(═O)(═O)O)cc2/N═N\c1ccc(Nc2ccccc2)c2c1cccc2S(═O)(═O)O iKeap59O═S(═O)(O)c1cc(/N═N\c2ccc(O)c3ccccc23)ccc1/C═C\c1ccc(/N═N\c2ccc(O)c3ccccc23)cc1S(═O)(═O)O iKeap58CC1═NN(c2ccc(S(═O)(═O)O)cc2)C(═O)[C@@H]1/N═N\c1ccc(-c2ccc(/N═N\c3ccc(O)c(C(═O)O)c3)c(C)c2)cc1C iKeap10Nc1ccc2cc(S(═O)(═O)O)cc(O)c2c1/N═N\c1ccc(-c2ccc(/N═N\c3c(N)ccc4cc(S(═O)(═O)O)cc(O)c43)cc2)cc1 iKeap71O═C(O)c1ccc(-c2ccc([C@H]3CC(═O)Oc4ccc5c(c43)O/C(═C\c3cc(F)c(F)c(F)c3)C5═O)o2)cc1 iKeap45Nc1ccc(/N═N\c2ccc(/C═C\c3ccc(/N═N\c4ccc(N)c5ccccc45)cc3S(═O)(═O)O)c(S(═O)(═O)O)c2)c2ccccc12 iKeap70COc1ccc(C(═O)N2CCC[C@H](C(═O)NNC(═O)c3ccc4ccccc4c3)C2)c2ccccc12 iKeap64O═C(Nc1ccc2[nH]c(-c3cccc(F)c3)nc2c1)[C@@H]1CCCN(C(═O)c2ccc3[nH]ncc3c2)C1 iKeap43O═C1c2ccccc2/C(═C\NNc2ccc(C(F)(F)F)cc2[N+](═O)[O−])C(═O)N1Cc1ccc2c(c1)OCO2 iKeap65CC1(C)Cc2oc3c(cc(NS(═O)(═O)c4ccc5c6c(cccc64)C(═O)N5)c4ccccc34) c2C(═O)C1iKeap44 O═C1c2ccccc2C(═O)N1Cc1cccc(C(═O)N2CCCC[C@H]2c2nc(-c3ccccc3)no2)c1 iKeap66O═S(═O)(Nc1cccc(-c2ccc3nnc(-c4cccnc4)n3n2)c1)c1ccc2ccccc2c1 iKeap48CC(═O)N/C(═C\c1ccc(Cc2nc3c([nH]2)C(═O)c2ccccc2C3═O)cc1)c1nc2c([nH]1)C(═O)c1ccccc1C2═O iKeap21O═C(NNc1nc2ccccn2n1)c1cc(-c2cccc3ccccc23)nc2ccccc12 iKeap15O═C(c1ccccc1)C1═C[C@@H]2[C@@H]3C(═O)N(c4cccc5ccccc54)C(═O)[C@@H]3[C@@H](C(═O)c3ccc(Br)cc3)N2C═C1 iKeap41O═C(c1cc(-c2ccc3c(c2)OCCO3)nc2ccccc21)N1CCC(Cc2ccccc2)CC1 iKeap76O═C(C1CCN(c2ccc3nnnn3n2)CC1)N1Cc2ccccc2-c2ccccc2C1 iKeap22O═c1cc(-c2ccc(O)cc2)oc2c1c(O)cc1c2[C@H](c2ccc3ncccc3c2)CC(═O)O1 iKeap73C[C@H]1CCc2c(sc3ncnc(N4CCO[C@@H](CN5C(═O)c6ccccc6C5═O)C4) c23)C1 iKeap74C[C@@H]1Oc2ccc(C(═O)C3CCN(C(═O)[C@H]4C[C@H]4c4cccc5ccccc45) CC3)cc2NC1═OiKeap8 O═C(Nc1cccc(-c2nnn/nH]2)c1)[C@@H]1C[C@H]2CCCC[C@H]2N1C(═O)c1ccc2ccccc2c1 iKeap33O═S(═O)(Nc1nc2ccccc2nc1N1CCC[C@@H](c2nc3ccccc3[nH]2)C1)c1ccccc1 iKeap61O═C(C1Cc2ccccc2C1)N1CCN(C(═O)C2Cc3ccccc3C2)c2ccccc21 iKeap49O═C1c2ccccc2C(═O)C(N/N═c2/[nH][nH]/c(═N\NC3═C(Cl)C(═O)c4ccccc4C3═O)c3ccccc32)═C1C1 iKeap19CC(═O)N5CCCC4═CC(NC(═O)c3cccc(NC(═O)C2Cc1ccccc1O2)c3)═CCC45 iKeap47O═C(OCc2cc([N+](═O)O)cc1COCOc12)c6c5CCC/C(═C/c4ccc3OCOc3c4) c5nc7ccccc67iKeap72 O═c3[nH]c2ccc(c1ccccc1)cc2c3═NNc6nc(c4ccccn4)nc5CCCc56

The compounds of the disclosure can inhibit KEAP1. Accordingly, anotheraspect of the disclosure provides a method of inhibiting KEAP1.Accordingly, in one aspect, provided herein is method for inhibitingKEAP1. Generally, the method comprises contacting KEAP1 with a compounddescribed herein. By inhibiting KEAP1 is meant inhibiting a biologicalactivity of KEAP1. For example, inhibiting its interaction with Nrf2.

The KEAP1 can be inside a cell whom contacted with a compound of thedisclosure. For example, a compound described herein can be administeredto a cell expressing KEAP1. It is noted that administering the compoundto the cell can be in vitro or in-vivo. Methods for administering acompound to a cell are well known and available to one of skill in theart. As used herein, administering the compound to the cell meanscontacting the cell with the compound so that the compound is taken upby the cell. Generally, the cell can be contacted with the compound in acell culture e.g., in vitro or ex vivo, or the compound can beadministrated to a subject, e.g., in vivo. The term “contacting” or“contact” as used herein in connection with contacting a cell includessubjecting the cells to an appropriate culture media, which comprises acompound described herein. Where the cell is in vivo, “contacting” or“contact” includes administering the compound, e.g., in a pharmaceuticalcomposition to a subject via an appropriate administration route suchthat the compound contacts the cell in vivo.

For example, when the cell is in vitro, said administering to the cellcan include subjecting the cell to an appropriate culture media whichcomprises the indicated compound. Where the cell is in vivo, saidadministering to the cell includes administering the compound to asubject via an appropriate administration route such that the compoundis administered to the cell in vivo.

The compounds of the disclosure can disrupt or inhibit binding of Nrf2to KEAP1, i.e., inhibit the KEAP1-NRF2 interaction. Accordingly, inanother aspect provided herein is a method for disrupting, preventing orinhibiting an interaction between KEAP1 and Nrf2. The method comprisescontacting KEAP1 with a compound described herein.

The compounds of the disclosure can inhibit the KEAP1-NRF2 interactionby more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 95%, 97%,or 99% of the positive control. In some embodiments, the compounds ofthe disclosure can inhibit the KEAP1-NRF2 interaction by more than about75%, 80%, 85%, 90%, 95%, 97%, or 99% of the positive control. Forexample, the compounds disclosed herein can inhibit the KEAP1-NRF2interaction by more than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the positive control.Furthermore, the compounds disclosed herein can inhibit the KEAP1-NRF2interaction with an IC₅₀ of less than about 3 mM, or less than about 2mM, or less than about 1 mM, or less than about 0.5 mM, or less thanabout 04 mM, or less than about 0.3 mM, or less than about 0.2 mM, orless than about 0.1 mM, or less than about 90 nM, or less than about 80nM, or less than about 70 nM, or less than about 60 nM, or less thanabout 50 nM, or less than about 40 nM, or less than about 30 nM, or lessthan about 20 nM, or less than about 10 nM, or less than about 5 nM.

Disrupting the interactions between KEAP1 and Nrf2 can activate theNrf2. Accordingly, in yet another aspect, provided herein is a methodfor activating Nrf2. The method comprises contacting KEAP1 with acompound described herein.

Certain embodiments provided herein relate to methods of inhibitingKEAP1, which can be useful for treating, preventing, or ameliorating adisease, disorder or condition associated with KEAP1 in a subject. Forexample, a disease, disorder or condition associated with dysfunction ofthe Nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-likeECH-associated protein 1 (KEAP1) axis such as a disease, disorder orcondition associated with Nrf2-KEAP1 interaction. Accordingly, in someembodiments of any one of the aspects, the subject has a disease,disorder or condition associated with dysfunction of the KEAP1-Nrf2axis. For example, a subject diagnosed with a disease, disorder orcondition associated with dysfunction of the KEAP1-Nrf2 axis.

Accordingly, in another aspect, provided herein is a method for treatinga disease, disorder or condition associated with KEAP1. Generally, themethod comprises administering a compound described herein to a subjectin need thereof.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a disease,disorder or condition associated with dysfunction of the Nrf2-KEAP1axis.

in some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a disease,disorder or condition associated with Nrf2-KEAP1 interaction.

Exemplary Diseases Associated with Dysfunction of KEAP1-Nrf2 Axis

An example of a disease, disorder or condition associated withdysfunction of KEAP1-Nrf2 axis is oxidative stress or a disease,disorder or condition associated with oxidative stress. Some exemplarydiseases, disorders or conditions associated with oxidative stressinclude but are not limited to, metabolic diseases, inflammatorydiseases, autoimmune diseases, lung diseases, cardiovascular diseases,liver diseases, kidney diseases, ophthalmological diseases,gastrointestinal tract diseases, neurological diseases,neurodegenerative diseases, and cancers.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a metabolicdisease. Exemplary metabolic diseases associated with KEAP1 include, butare not limited to, metabolic syndrome, type 2 diabetes, diabeticnephropathy, diabetic cardiomyopathy and insulin resistance.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a liver disease.Exemplary diseases, disorder or conditions of the liver associated withKEAP1 include, but are not limited to, hepatic fibrosis, autosomaldominant polycystic liver disease, hepatic steatosis, non-alcoholicsteatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD).

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is an inflammatorydisease. Exemplary inflammatory diseases, disorder or conditionsassociated with KEAP1 include, but are not limited to, asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and airwayhyperresponsiveness.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is an autoimmunedisease. Exemplary autoimmune diseases, disorder or conditionsassociated with KEAP1 include, but are not limited to, multiplesclerosis, psoriasis, connective tissue disease, and pulmonary arterialhypertension associated with connective tissue disease.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a kidneydisease. Exemplary kidney diseases, disorder or conditions associatedwith KEAP1 include, but are not limited to, renal fibrosis, Alportsyndrome, autosomal dominant polycystic kidney disease, chronic kidneydisease, IgA nephropathy, type 1 diabetes, type 2 diabetes mellitus, andfocal segmental glomerulosclerosis, and nephropathy.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a lung disease.Exemplary lung diseases, disorder or conditions associated with KEAP1include, but are not limited to, pulmonary arterial hypertension,pulmonary hypertension-interstitial lung disease, pulmonary fibrosis,cystic fibrosis, emphysema, chronic obstructive pulmonary disease(COPD), and chronic bronchitis.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a cardiovasculardisease. Exemplary cardiovascular diseases, disorder or conditionsassociated with KEAP1 include, but are not limited to, atherosclerosis,heart failure, myocardial infarction, reperfusion injury, and stroke.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is a neurologicalor neurodegenerative disease. Exemplary neurological orneurodegenerative diseases, disorder or conditions associated with KEAP1include, but are not limited to, Friedreich's ataxia, subarachnoidhemorrhage, amyotrophic lateral sclerosis, Parkinson's disease,Parkinson's disease with dementia with Lewy body, Huntington's Disease,Batten Disease, multiple system atrophy (MSA), progressive supranuclearpalsy (PSA), corticobasal degeneration (CBD), frontotemporal lobedegeneration, Alzheimer's disease, Fragile X syndrome, chronic fatiguesyndrome, cerebral ischemia, neuronal cell death, Creutzfeldt-Jakobdisease, Lewy body disease, Pick's disease, or neurofibromatosis.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is anophthalmological disease. Exemplary ophthalmological diseases, disorderor conditions associated with KEAP1 include, but are not limited to, isdry eye macular degeneration, retinovascular disease, or retinopathy.

In some embodiments of any one of the aspects described herein, thedisease, disorder or condition associated with KEAP1 is cancer. It isnoted that a cancer associated with KEAP1 can breast cancer, livercancer, lung cancer, breast cancer, prostate cancer, colon cancer,neuroblastoma, or leukemia.

The disease, disorder or condition associated with dysfunction of theKEAP1-Nrf2 axis can be selected from the group consisting of Alportsyndrome, amyotrophic lateral sclerosis, autosomal dominant polycystickidney disease, bone disease, blood disease, chronic kidney disease,chronic obstructive pulmonary disease, connective tissue disease, dryeye macular degeneration, estrogen receptor-positive breast cancer, eyedisease, focal segmental glomerulosclerosis, Friedreich ataxia,immunoglobulin A nephropathy, interstitial lung disease, lung diseases,multiple sclerosis, kidney disease, neurodegenerative disease, primaryfocal segmental glomerulosclerosis, psoriasis, pulmonary arterialhypertension, retinovascular disease, subarachnoid hemorrhage, type 1diabetes, and type 2 diabetes mellitus. In various embodiments, thepreclinical disease or disorder is selected from the group consistingof, autoimmune diseases (e.g., rheumatoid arthritis, Sjogren syndrome,STING-dependent interferonopathies, systemic lupus erythematous,vitiligo); respiratory diseases (e.g., chronic obstructive pulmonarydisease, chronic sarcoidosis, emphysema, hypersensitivity pneumonitis,idiopathic pulmonary fibrosis, pulmonary fibrosis); gastrointestinaldiseases (e.g., hemochromatosis, hepatic fibrosis, primary biliarycholangitis and cirrhosis); metabolic diseases (e.g., insulinresistance, glomerulonephritis, nonalcoholic steatohepatitis, type 2diabetes mellitus, vascular dysfunction); cardiovascular diseases (e.g.,atherosclerosis, diabetic vascular disease, hypertension, myocardialischemia-reperfusion injury, heart failure); neurodegenerative diseases(e.g., Alzheimer disease, Fluntington disease, Friedrich ataxia,Parkinson disease); skin diseases (e.g., chronic/diabetic woundhealing); and cancer.

Routes of Administration

It is noted that the terms “administered” and “subjected” are usedinterchangeably in the context of treatment of a disease or disorder. Injurisdictions that forbid the patenting of methods that are practiced onthe human body, the meaning of “administering” of a composition to ahuman subject shall be restricted to prescribing a controlled substancethat a human subject will be administer to the subject by any technique(e.g., orally, inhalation, topical application, injection, insertion,etc.). The broadest reasonable interpretation that is consistent withlaws or regulations defining patentable subject matter is intended. Injurisdictions that do not forbid the patenting of methods that arepracticed on the human body, the “administering” of compositionsincludes both methods practiced on the human body and also the foregoingactivities.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. In someembodiments, administration will generally be local rather thansystemic.

In some embodiments, a compound of the disclosure is orallyadministered. Without limitations, oral administration can be in theform of solutions, suspensions, tablets, pills, capsules,sustained-release formulations, oral rinses, powders and the like.

In some embodiments, a compound of the disclosure is compound isadministered in a local rather than systemic manner, for example, viatopical application of the compound directly on to skin, orintravenously, or subcutaneously, often in a depot preparation orsustained release formulation. In specific embodiments, long actingformulations are administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. In yetother embodiments, the compound as described herein is provided in theform of a rapid release formulation, in the form of an extended releaseformulation, or in the form of an intermediate release formulation. Inyet other embodiments, the compound described herein is administeredtopically (e.g., as a patch, an ointment, or in combination with a wounddressing, or as a wash or a spray). In alternative embodiments, aformulation is administered systemically (e.g., by injection, or as apill).

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compounddescribed herein which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells, e.g., inhibitKEAP1 in a subject at a reasonable benefit/risk ratio applicable to anymedical treatment. Thus, “therapeutically effective amount” means thatamount which, when administered to a subject for treating a disease, issufficient to affect such treatment for the disease.

Depending on the route of administration, effective doses can becalculated according to the body weight, body surface area, or organsize of the subject to be treated. Optimization of the appropriatedosages can readily be made by one skilled in the art in light ofpharmacokinetic data observed in human clinical trials. Alternatively,or additionally, the dosage to be administered can be determined fromstudies using animal models for the particular type of condition to betreated, and/or from animal or human data obtained from agents which areknown to exhibit similar pharmacological activities. The final dosageregimen will be determined by the attending surgeon or physician,considering various factors which modify the action of active agent,e.g., the agent's specific activity, the agent's specific half-life invivo, the severity of the condition and the responsiveness of thepatient, the age, condition, body weight, sex and diet of the patient,the severity of any present infection, time of administration, the use(or not) of other concomitant therapies, and other clinical factors.

Determination of an effective amount is well within the capability ofthose skilled in the art. Generally, the actual effective amount canvary with the specific compound, the use or application technique, thedesired effect, the duration of the effect and side effects, thesubject's history, age, condition, sex, as well as the severity and typeof the medical condition in the subject, and administration of otherpharmaceutically active agents. Accordingly, an effective dose ofcompound described herein is an amount sufficient to produce at leastsome desired therapeutic effect in a subject.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of use or administration utilized.

The effective dose can be estimated initially from cell culture assays.A dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay. The effective plasma concentration for a compound as disclosedherein can be about 0.01 μM to about 10 μM, about 0.2 μM to about 5 μM,or about 0.8 to about 3 μM in a subject, such as a rat, dog, or human.

Generally, the compositions are administered so that a compound of thedisclosure herein is used or given at a dose from 1 μg/kg to 1000 mg/kg;1 μg/kg to 500 mg/kg; 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to1 mg/kg, 100 μg/kg to 100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20mg/kg, 100 μg/kg to 10 mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It isto be understood that ranges given here include all intermediate ranges,for example, the range 1 mg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg,1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6mg/kg, 1 mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to10 mg/kg, 6 mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg,9 mg/kg to 10 mg/kg, and the like. Further contemplated is a dose(either as a bolus or continuous infusion) of about 0.1 mg/kg to about10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, or 0.5 mg/kg to about 3mg/kg. It is to be further understood that the ranges intermediate tothose given above are also within the scope of this disclosure, forexample, in the range 1 mg/kg to 10 mg/kg, for example use or doseranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6mg/kg, and the like.

The compounds described herein can be administered at once, or can bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment will be a function of the location of where the composition isparenterally administered, the carrier and other variables that can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values can also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens can need to be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of theformulations. Hence, the concentration ranges set forth herein areintended to be exemplary and are not intended to limit the scope orpractice of the claimed formulations.

The compound can be administered as a single bolus or multiple boluses,as a continuous infusion, or a combination thereof. For example, thecompound can be administered as a single bolus initially, and thenadministered as a continuous infusion following the bolus. The rate ofthe infusion can be any rate sufficient to maintain effectiveconcentration, for example, to maintain effective plasma concentration.Some contemplated infusion rates include from 1 μg/kg/min to 100mg/kg/min, or from 1 μg/kg/hr to 1000 mg/kg/hr. Rates of infusion caninclude 0.2 to 1.5 mg/kg/min, or more specifically 0.25 to 1 mg/kg/min,or even more specifically 0.25 to 0.5 mg/kg/min. It will be appreciatedthat the rate of infusion can be determined based upon the dosenecessary to maintain effective plasma concentration and the rate ofelimination of the compound, such that the compound is administered viainfusion at a rate sufficient to safely maintain a sufficient effectiveplasma concentration of compound in the bloodstream.

Pharmaceutical Compositions/Formulations

For administration to a subject, the compounds describe herein can beprovided in a pharmaceutically acceptable compositions. Thesepharmaceutically acceptable compositions comprise a compound describedherein, formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions described herein can be specially formulatedfor administration in solid or liquid form, including those adapted forthe following: (1) oral administration, for example, drenches (aqueousor non-aqueous solutions or suspensions), gavages, lozenges, dragees,capsules, pills, tablets (e.g., those targeted for buccal, sublingual,and systemic absorption), boluses, powders, granules, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular, intravenous or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation; (3) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; or (9) nasally. Additionally, compounds can be implantedinto a patient or injected using a drug delivery system. See, forexample, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which isherein incorporated by reference.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-Cu alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

Examples of solid carriers include starch, sugar, bentonite, silica, andother commonly used carriers. Further non-limiting examples of carriersand diluents which can be used in the formulations comprising a compounddescribed herein as disclosed herein of the present invention includesaline, syrup, dextrose, and water.

Pharmaceutically-acceptable antioxidants include, but are not limitedto, (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acids, and the like.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition which is effective forproducing some desired therapeutic effect in at least a sub-populationof cells in an animal at a reasonable benefit/risk ratio applicable toany medical treatment. According, a “therapeutically effective amount”refers to an amount effective, at dosage and periods of time necessary,to achieve a desired therapeutic result. A therapeutic result can be,e.g., lessening of symptoms, prolonged survival, improved mobility, andthe like. A therapeutic result need not be a “cure.”

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

The compounds can be formulated in a gelatin capsule, in tablet form,dragee, syrup, suspension, topical cream, suppository, injectablesolution, or kits for the preparation of syrups, suspension, topicalcream, suppository or injectable solution just prior to use. Also,compounds can be included in composites, which facilitate its slowrelease into the blood stream, e.g., silicon disc, polymer beads.

The formulations can conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Techniques, excipients and formulations generally are found in, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1985, 17th edition, Nema et al., PDA J Pharm. Sci. Tech. 199751:166-171. Methods to make invention formulations include the step ofbringing into association or contacting an ActRIIB compound with one ormore excipients or carriers. In general, the formulations are preparedby uniformly and intimately bringing into association one or morecompounds with liquid excipients or finely divided solid excipients orboth, and then, if appropriate, shaping the product.

The preparative procedure may include the sterilization of thepharmaceutical preparations. The compounds may be mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, salts forinfluencing osmotic pressure, etc., which do not react deleteriouslywith the compounds.

Examples of injectable form include solutions, suspensions andemulsions. Injectable forms also include sterile powders forextemporaneous preparation of injectable solutions, suspensions oremulsions. The compounds of the present invention can be injected inassociation with a pharmaceutical carrier such as normal saline,physiological saline, bacteriostatic water, Cremophor™ EL (BASF,Parsippany, N.J.), phosphate buffered saline (PBS), Ringer's solution,dextrose solution, ethanol, polyol (e.g., glycerol, propylene glycol,and liquid polyethylene glycol), vegetable oils, and suitable mixturesthereof, and other aqueous carriers known in the art. Appropriatenon-aqueous carriers may also be used and examples include fixed oilsand ethyl oleate. In all cases, the composition must be sterile andshould be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin. A suitablecarrier is 5% dextrose in saline. Frequently, it is desirable to includeadditives in the carrier such as buffers and preservatives or othersubstances to enhance isotonicity and chemical stability.

In some embodiments, compounds can be administrated encapsulated withinliposomes. The manufacture of such liposomes and insertion of moleculesinto such liposomes being well known in the art, for example, asdescribed in U.S. Pat. No. 4,522,811. Liposomal suspensions (includingliposomes targeted to particular cells, e.g., a pituitary cell) can alsobe used as pharmaceutically acceptable carriers.

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the composition can be administered in asustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185; content ofeach of which is incorporated herein by reference. These dosage formscan be used to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

In some embodiments, the compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc.

In the case of oral ingestion, excipients useful for solid preparationsfor oral administration are those generally used in the art, and theuseful examples are excipients such as lactose, sucrose, sodiumchloride, starches, calcium carbonate, kaolin, crystalline cellulose,methyl cellulose, glycerin, sodium alginate, gum arabic and the like,binders such as polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, ethyl cellulose, gum arabic, shellac, sucrose, water,ethanol, propanol, carboxymethyl cellulose, potassium phosphate and thelike, lubricants such as magnesium stearate, talc and the like, andfurther include additives such as usual known coloring agents,disintegrators such as alginic acid and Primogel™, and the like. Thecompounds can be orally administered, for example, with an inertdiluent, or with an assimilable edible carrier, or they may be enclosedin hard or soft shell capsules, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, these compounds may be incorporated withexcipients and used in the form of tablets, capsules, elixirs,suspensions, syrups, and the like. Such compositions and preparationsshould contain at least 0.1% of compound. The percentage of the agent inthese compositions may, of course, be varied and may conveniently bebetween about 2% to about 60% of the weight of the unit. The amount ofcompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions according tothe present invention are prepared so that an oral dosage unit containsbetween about 100 and 2000 mg of compound. Examples of bases useful forformulation of suppositories are oleaginous bases such as cacao butter,polyethylene glycol, lanolin, fatty acid triglycerides, witepsol(trademark, Dynamite Nobel Co. Ltd.) and the like. Liquid preparationsmay be in the form of aqueous or oleaginous suspension, solution, syrup,elixir and the like, which can be prepared by a conventional way usingadditives. The compositions can be given as a bolus dose, to maximizethe circulating levels for the greatest length of time after the dose.Continuous infusion may also be used after the bolus dose.

The compounds can also be administrated directly to the airways in theform of an aerosol. For administration by inhalation, the compounds insolution or suspension can be delivered in the form of an aerosol sprayfrom pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or hydrocarbonpropellant like propane, butane or isobutene. The compounds can also beadministrated in a no-pressurized form such as in an atomizer ornebulizer.

In the case of a pressurized aerosol, the dosage unit may be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, such as, by way of example only, gelatin for use in aninhaler or insufflator may be formulated containing a powder mix of thecompound described herein and a suitable powder base such as lactose orstarch.

Representative intranasal formulations are described in, for example,U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Formulations thatinclude a compound described herein are prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, fluorocarbons,and/or other solubilizing or dispersing agents known in the art. See,for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are known to those skilled inthe preparation of nasal dosage forms and some of these can be found inREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005. Thechoice of suitable carriers is dependent upon the exact nature of thenasal dosage form desired, e.g., solutions, suspensions, ointments, orgels. Nasal dosage forms generally contain large amounts of water inaddition to the active ingredient. Minor amounts of other ingredientssuch as pH adjusters, emulsifiers or dispersing agents, preservatives,surfactants, gelling agents, or buffering and other stabilizing andsolubilizing agents are optionally present. Preferably, the nasal dosageform should be isotonic with nasal secretions

The compounds can also be administered parenterally. Solutions orsuspensions of these compounds can be prepared in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof in oils. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

It may be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage. Asused herein, “dosage unit” refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

Administration can also be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the compounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

For oral or enteral formulations as disclosed herein for use with thepresent invention, tablets can be formulated in accordance withconventional procedures employing solid carriers well-known in the art.Capsules employed for oral formulations to be used with the methods ofthe present invention can be made from any pharmaceutically acceptablematerial, such as gelatin or cellulose derivatives. Sustained releaseoral delivery systems and/or enteric coatings for orally administereddosage forms are also contemplated, such as those described in U.S. Pat.No. 4,704,295, “Enteric Film-Coating Compositions,” issued Nov. 3, 1987;U.S. Pat. No. 4,556,552, “Enteric Film-Coating Compositions,” issuedDec. 3, 1985; U.S. Pat. No. 4,309,404, “Sustained Release PharmaceuticalCompositions,” issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406,“Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982.

Also provided herein is a tablet formulation comprising a compounddescribed herein with an enteric polymer casing. An example of such apreparation can be found in WO2005/021002. The active material in thecore can be present in a micronised or solubilised form. In addition toactive materials the core can contain additives conventional to the artof compressed tablets. Appropriate additives in such a tablet cancomprise diluents such as anhydrous lactose, lactose monohydrate,calcium carbonate, magnesium carbonate, dicalcium phosphate or mixturesthereof; binders such as microcrystalline cellulose,hydroxypropylmethylcellulose, hydroxypropyl-cellulose,polyvinylpyrrolidone, pre-gelatinised starch or gum acacia or mixturesthereof, disintegrants such as microcrystalline cellulose (fulfillingboth binder and disintegrant functions) cross-linkedpolyvinylpyrrolidone, sodium starch glycollate, croscarmellose sodium ormixtures thereof, lubricants, such as magnesium stearate or stearicacid, glidants or flow aids, such as colloidal silica, talc or starch,and stabilisers such as desiccating amorphous silica, colouring agents,flavours etc. Preferably the tablet comprises lactose as diluent. When abinder is present, it is preferably hydroxypropylmethyl cellulose.Preferably, the tablet comprises magnesium stearate as lubricant.Preferably the tablet comprises croscarmellose sodium as disintegrant.Preferably, the tablet comprises microcrystalline cellulose.

The diluent can be present in a range of 10-80% by weight of the core.The lubricant can be present in a range of 0.25-2% by weight of thecore. The disintegrant can be present in a range of 1-10% by weight ofthe core. Microcrystalline cellulose, if present, can be present in arange of 10-80% by weight of the core.

The active ingredient, e.g., a compound described herein preferablycomprises between 10 and 50% of the weight of the core, more preferablybetween 15 and 35% of the weight of the core (calculated as free baseequivalent). The core can contain any therapeutically suitable dosagelevel of the active ingredient, but preferably contains up to 150 mg ofthe active ingredient. Particularly preferably, the core contains 20,30, 40, 50, 60, 80 or 100 mg of the active ingredient. The activeingredient can be present as is or as any pharmaceutically acceptablesalt. If the active ingredient is present as a salt, the weight isadjusted such that the tablet contains the desired amount of activeingredient, calculated as free base or free acid of the salt.

The core can be made from a compacted mixture of its components. Thecomponents can be directly compressed, or can be granulated beforecompression. Such granules can be formed by a conventional granulatingprocess as known in the art. In an alternative embodiment, the granulescan be individually coated with an enteric casing, and then enclosed ina standard capsule casing.

The core is surrounded by a casing which comprises an enteric polymer.Examples of enteric polymers are cellulose acetate phthalate, celluloseacetate succinate, methylcellulose phthalate, ethylhydroxycellulosephthalate, polyvinylacetate phthalate, polyvinylbutyrate acetate, vinylacetate-maleic anhydride copolymer, styrene-maleic mono-ester copolymer,methyl acrylate-methacrylic acid copolymer or methacrylate-methacrylicacid-octyl acrylate copolymer. These can be used either alone or incombination, or together with other polymers than those mentioned above.The casing can also include insoluble substances which are neitherdecomposed nor solubilised in living bodies, such as alkyl cellulosederivatives such as ethyl cellulose, crosslinked polymers such asstyrene-divinylbenzene copolymer, polysaccharides having hydroxyl groupssuch as dextran, cellulose derivatives which are treated withbifunctional crosslinking agents such as epichlorohydrin, dichlorohydrinor 1, 2-, 3, 4-diepoxybutane. The casing can also include starch and/ordextrin.

In some embodiments, an enteric coating materials are the commerciallyavailable Eudragit® enteric polymers such as Eudragit® L, Eudragit® Sand Eudragit® NE used alone or with a plasticiser. Such coatings arenormally applied using a liquid medium, and the nature of theplasticiser depends upon whether the medium is aqueous or non-aqueous.Plasticisers for use with aqueous medium include propylene glycol,triethyl citrate, acetyl triethyl citrate or Citroflex® or Citroflex®A2. Non-aqueous plasticisers include these, and also diethyl and dibutylphthalate and dibutyl sebacate. A preferred plasticiser is Triethylcitrate. The quantity of plasticiser included will be apparent to thoseskilled in the art.

The casing can also include an anti-tack agent such as talc, silica orglyceryl monostearate. Preferably the anti-tack agent is glycerylmonostearate. Typically, the casing can include around 5-25 wt %Plasticizers and up to around 50 wt % of anti-tack agent, preferably1-10 wt % of anti-tack agent.

If desired, a surfactant can be included to aid with forming an aqueoussuspension of the polymer. Many examples of possible surfactants areknown to the person skilled in the art. Preferred examples ofsurfactants are polysorbate 80, polysorbate 20, or sodium laurylsulphate. If present, a surfactant can form 0.1-10% of the casing,preferably 0.2-5% and particularly preferably 0.5-2%.

A seal coat can also be included between the core and the entericcoating. A seal coat is a coating material which can be used to protectthe enteric casing from possible chemical attack by any alkalineingredients in the core. The seal coat can also provide a smoothersurface, thereby allowing easier attachment of the enteric casing. Aperson skilled in the art would be aware of suitable coatings.Preferably the seal coat is made of an Opadry coating, and particularlypreferably it is Opadry White OY-S-28876. Other enteric-coatedpreparations of this sort can be prepared by one skilled in the art,using these materials or their equivalents.

For intravenous injections or drips or infusions, compounds describedherein are formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart. For other parenteral injections, appropriate formulations includeaqueous or nonaqueous solutions, preferably with physiologicallycompatible buffers or excipients. Such excipients are known.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspension,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. In one aspect, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Some exemplary embodiments of various aspects can be described by thefollowing numbered embodiments.

Embodiment 1: A method of treating a disease associated with dysfunctionof the Nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-likeECH-associated protein 1 (KEAP1) axis in a subject in need thereof, themethod comprising administering to the subject a compound of Formula(I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted arylene, substituted or        unsubstituted biarylene, or substituted or unsubstituted        heteroarylene;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 2: A method of treating a disease associated with Nrf2-KEAP1interaction, the method comprising administering to a subject in needthereof a therapeutically effective amount of a compound of Formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or        a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 3: The method of Embodiment 1 or 2, wherein the disease isassociated with oxidative stress.

Embodiment 4: The method of any of Embodiments 1-3, wherein the diseaseis a metabolic disease, an inflammatory disease, an autoimmune disease,a lung disease, a cardiovascular disease, a liver disease, a kidneydisease, an ophthalmological disease, a gastrointestinal tract disease,a neurological disease, a neurodegenerative disease, or cancer.

Embodiment 5: The method of any of Embodiments 1-4, wherein the diseaseis a metabolic disease.

Embodiment 6: The method of any of Embodiments 1-5, wherein the diseaseis metabolic syndrome, type 2 diabetes, diabetic nephropathy, diabeticcardiopathy, obesity, or insulin resistance.

Embodiment 7: The method of any of Embodiments 1-4, wherein the diseaseis a liver disease.

Embodiment 8: The method of any of Embodiments 1-4 or 7, wherein thedisease is hepatic fibrosis, autosomal dominant polycystic liverdisease, hepatic steatosis, non-alcoholic steatohepatitis (NASH), ornon-alcoholic fatty liver disease (NAFLD).

Embodiment 9: The method of any of Embodiments 1-4, wherein the diseaseis an inflammatory disease.

Embodiment 10: The method of any of Embodiments 1-4 or 9, wherein thedisease is asthma, chronic obstructive pulmonary disease (COPD), cysticfibrosis, or airway hyperresponsiveness.

Embodiment 11: The method of any of Embodiments 1-4, wherein the diseaseis an autoimmune disease.

Embodiment 12: The method of any of Embodiments 1-4 or 11, wherein thedisease is multiple sclerosis, psoriasis, connective tissue disease, orpulmonary arterial hypertension associated with connective tissuedisease.

Embodiment 13: The method of any of Embodiments 1-4, wherein the diseaseis a kidney disease.

Embodiment 14: The method of any of Embodiments 1-4 or 13, wherein thedisease is renal fibrosis, Alport syndrome, autosomal dominantpolycystic kidney disease, chronic kidney disease, IgA nephropathy, type1 diabetes, type 2 diabetes mellitus, and focal segmentalglomerulosclerosis, or nephropathy.

Embodiment 15: The method of any of Embodiments 1-4, wherein the diseaseis a lung disease.

Embodiment 16: The method of any of Embodiments 1-4 or 15, wherein thedisease is pulmonary arterial hypertension, pulmonaryhypertension-interstitial lung disease, pulmonary fibrosis, cysticfibrosis, emphysema, chronic obstructive pulmonary disease (COPD), orchronic bronchitis.

Embodiment 17: The method of any of Embodiments 1-4, wherein the diseaseis a cardiovascular disease.

Embodiment 18: The method of any of Embodiments 1-4 or 17, wherein thedisease is atherosclerosis, heart failure, myocardial infarction,reperfusion injury, or stroke.

Embodiment 19: The method of any of Embodiments 1-4, wherein the diseaseis a neurological or neurodegenerative disease.

Embodiment 20: The method of any of Embodiments 1-4 or 19, wherein thedisease is Friedreich's ataxia, subarachnoid hemorrhage, amyotrophiclateral sclerosis, Parkinson's disease, Parkinson's disease withdementia with Lewy body, Huntington's Disease, Batten Disease, multiplesystem atrophy (MSA), progressive supranuclear palsy (PSA), corticobasaldegeneration (CBD), frontotemporal lobe degeneration, Alzheimer'sdisease, Fragile X syndrome, chronic fatigue syndrome, cerebralischemia, neuronal cell death, Creutzfeldt-Jakob disease, Lewy bodydisease, Pick's disease, or neurofibromatosis.

Embodiment 21: The method of any of Embodiments 1-4, wherein the diseaseis an ophthalmological disease.

Embodiment 22: The method of any of Embodiments 1-4 or 21, wherein thedisease is dry eye macular degeneration, retinovascular disease, orretinopathy.

Embodiment 23: The method of any of Embodiments 1-4, wherein the diseaseis cancer.

Embodiment 24: The method of any of Embodiments 1-4 or 23, wherein thedisease is breast cancer, liver cancer, lung cancer, breast cancer,prostate cancer, colon cancer, neuroblastoma, or leukemia.

Embodiment 25: The method of any of Embodiments 1-24, wherein thesubject is a human.

Embodiment 26: The method of any of Embodiments 1-25, wherein thecompound is of Formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 27: The method of any of Embodiments 1-26, wherein thecompound is of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 28: The method of any of Embodiments 1-25, wherein thecompound is of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 29: The method of any of Embodiments 1-25 or 28, wherein thecompound is selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 30: The method of any of Embodiments 1-25, wherein thecompound is of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 31: The method of any of Embodiments 1-25 or 30, wherein thecompound is of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 32: The method of any of Embodiments 1-25, wherein thecompound is of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 33: The method of any of Embodiments 1-25, wherein thecompound is of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 34: A method of inhibiting KEAP1, the method comprisingcontacting KEAP1 with a compound of the formula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 35: A method of disrupting the interaction between KEAP1 andNrf2, the method comprising contacting KEAP1 with a compound of Formula(I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or        a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound of Formula (III)

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 36: A method of preventing or inhibiting the interactionbetween KEAP1 and Nrf2, the method comprising contacting KEAP1 with acompound of Formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR, or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or        a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 37: A method of activating Nrf2, the method comprisingcontacting KEAP1 with a compound of Formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or        a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Embodiment 38: A pharmaceutical composition comprising a compound of theformula:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl;    -   R² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R³ is hydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂;    -   each R^(A) independently is H, substituted or unsubstituted        alkyl, substituted or unsubstituted alkenyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted heterocyclyl, or substituted or        unsubstituted cyclyl; or        a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   A is substituted or unsubstituted aryl, substituted or        unsubstituted biaryl, or substituted or unsubstituted        heteroaryl;    -   R⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁵ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound of Formula (III):

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof, wherein:

-   -   each A is independently substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; and    -   R⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, or substituted or unsubstituted        heterocyclyl; or        a compound selected from the following:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.

Some Selected Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%,±4%, ±4.5%, or ±5%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

As used herein the terms “comprising” or “comprises” means “including”or “includes” and are used in reference to compositions, methods,systems, and respective component(s) thereof, that are useful to theinvention, yet open to the inclusion of unspecified elements, whetheruseful or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, systems, andrespective components thereof as described herein, which are exclusiveof any element not recited in that description of the embodiment.

The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” are used herein to characterize a method or process thatis aimed at (1) delaying or preventing the onset of a disease orcondition; (2) slowing down or stopping the progression, aggravation, ordeterioration of the symptoms of the disease or condition; (3) bringingabout ameliorations of the symptoms of the disease or condition; or (4)curing the disease or condition. The term “treating” includes reducingor alleviating at least one adverse effect or symptom of a condition,disease or disorder. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted. Thatis, “treatment” includes not just the improvement of symptoms ormarkers, but also slowing of, progress or worsening of symptoms comparedto what would be expected in the absence of treatment. Beneficial ordesired clinical results include, but are not limited to, alleviation ofone or more symptom(s), diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, remission(whether partial or total), and/or decreased morbidity or mortality. Theterm “treatment” of a disease also includes providing relief from thesymptoms or side-effects of the disease (including palliativetreatment). A treatment can be administered prior to the onset of thedisease, for a prophylactic or preventive action. Alternatively, oradditionally, the treatment can be administered after initiation of thedisease or condition, for a therapeutic action.

In some embodiments, treatment is therapeutic and does not includeprophylactic treatment.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time. The particular combination oftherapies (therapeutics or procedures) to employ in such a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved.

As used herein, the term “subject” refers to any living organism whichcan be administered compound and/or pharmaceutical compositions of thepresent invention. The term includes, but is not limited to, humans,non-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses,domestic subjects such as dogs and cats, laboratory animals includingrodents such as mice, rats and guinea pigs, and the like. The term doesnot denote a particular age or sex. Thus, adult, child and newbornsubjects, whether male or female, are intended to be covered. The term“subject” is also intended to include living organisms susceptible toconditions or disease states as generally disclosed, but not limited to,throughout this specification. Examples of subjects include humans,dogs, cats, cows, goats, and mice. The term subject is further intendedto include transgenic species. The term “subject” and “individual” areused interchangeably herein, and refer to an animal, for example a humanor non-human mammals/animals, to whom treatment, including prophylactictreatment, with the compounds and compositions according to the presentinvention, is provided. The term “non-human animals” and “non-humanmammals” are used interchangeably herein and include all vertebrates,e.g., mammals, such as non-human primates, (particularly higherprimates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat,pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians,reptiles etc.

In some embodiments, the subject is a human or animal. Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomolgous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient” and “subject” areused interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of diseasesor disorders associated with dysfunction of Nrf2/KEAP1 axis.

It is noted that a human subject can be of any age, gender, race orethnic group, e.g., Caucasian (white), Asian, African, black, AfricanAmerican, African European, Hispanic, Middle eastern, etc.

In addition, the methods described herein can be used to treatdomesticated animals and/or pets. A subject can be male or female. Asubject can be one who has been previously diagnosed with or identifiedas suffering from or having a disease or disorder associated withdysfunction of Nrf2/KEAP1 axis, but need not have already undergonetreatment.

In some embodiments of any one of the aspects, the subject is human.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbongroup which can be straight or branched having 1 to about 60 carbonatoms in the chain, and which preferably have about 6 to about 50carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 toabout 8 carbon atoms. “Higher alkyl” refers to an alkyl group havingabout 10 to about 20 carbon atoms. The alkyl group can be optionallysubstituted with one or more alkyl group substituents which can be thesame or different, where “alkyl group substituent” includes halo, amino,aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio,aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain.Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl,t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl,tetradecyl, pentadecyl and hexadecyl. Useful alkyl groups includebranched or straight chain alkyl groups of 6 to 50 carbon, and alsoinclude the lower alkyl groups of 1 to about 4 carbons and the higheralkyl groups of about 12 to about 16 carbons.

A “heteroalkyl” group substitutes any one of the carbons of the alkylgroup with a heteroatom having the appropriate number of hydrogen atomsattached (e.g., a CH₂ group to an NH group or an O group). The term“heteroalkyl” include optionally substituted alkyl, alkenyl and alkynylradicals which have one or more skeletal chain atoms selected from anatom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus,silicon, or combinations thereof. In certain embodiments, theheteroatom(s) is placed at any interior position of the heteroalkylgroup. Examples include, but are not limited to, —CH₂—O—CH₃,—CH₂—CH₂—O—CH₃, —CH₂—NH—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—N(CH₃)—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. In some embodiments, up to two heteroatoms areconsecutive, such as, by way of example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃

As used herein, the term “alkenyl” refers to an alkyl group containingat least one carbon-carbon double bond. The alkenyl group can beoptionally substituted with one or more “alkyl group substituents.”Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl,dodecenyl, tetradecadienyl, heptadec-8-en-1-yl andheptadec-8,11-dien-1-yl.

As used herein, the term “alkynyl” refers to an alkyl group containing acarbon-carbon triple bond. The alkynyl group can be optionallysubstituted with one or more “alkyl group substituents.” Exemplaryalkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl anddodecynyl. Useful alkynyl groups include the lower alkynyl groups.

As used herein, the term “cycloalkyl” refers to a non-aromatic mono- ormulticyclic ring system of about 3 to about 12 carbon atoms. Thecycloalkyl group can be optionally partially unsaturated. The cycloalkylgroup can be also optionally substituted with an aryl group substituent,oxo and/or alkylene. Representative monocyclic cycloalkyl rings includecyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkylrings include adamantyl, octahydronaphthyl, decalin, camphor, camphane,and noradamantyl.

“Heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic, 8-12membered bicyclic, or 11-14 membered tricyclic ring system having 1-3heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). C_(x)heterocyclyl andC_(x)-C_(y)heterocyclyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. In some embodiments, 1, 2 or3 hydrogen atoms of each ring can be substituted by a substituent.Exemplary heterocyclyl groups include, but are not limited topiperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl,piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl,perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl,1,4-dioxanyland the like.

“Aryl” refers to an aromatic carbocyclic radical containing about 3 toabout 13 carbon atoms. The aryl group can be optionally substituted withone or more aryl group substituents, which can be the same or different,where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl,aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo,nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl,alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and—NRR′, where R and R′ are each independently hydrogen, alkyl, aryl andaralkyl. Exemplary aryl groups include substituted or unsubstitutedphenyl and substituted or unsubstituted naphthyl.

“Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12membered fused bicyclic, or 11-14 membered fused tricyclic ring systemhaving 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively.

Exemplary aryl and heteroaryls include, but are not limited to, phenyl,pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl,pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl,benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl,naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl and xanthenyl, and the like. In someembodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can besubstituted by a substituent.

As used herein, the term “halogen” or “halo” refers to an atom selectedfrom fluorine, chlorine, bromine and iodine. The term “halogenradioisotope” or “halo isotope” refers to a radionuclide of an atomselected from fluorine, chlorine, bromine and iodine.

A “halogen-substituted moiety” or “halo-substituted moiety”, as anisolated group or part of a larger group, means an aliphatic, alicyclic,or aromatic moiety, as described herein, substituted by one or more“halo” atoms, as such terms are defined in this application.

The term “haloalkyl” as used herein refers to alkyl and alkoxystructures structure with at least one substituent of fluorine, chorine,bromine or iodine, or with combinations thereof. In embodiments, wheremore than one halogen is included in the group, the halogens are thesame or they are different. The terms “fluoroalkyl” and “fluoroalkoxy”include haloalkyl and haloalkoxy groups, respectively, in which the halois fluorine. Exemplary halo-substituted alkyl includes haloalkyl,dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g.halosubstituted (C₁-C₃)alkyl includes chloromethyl, dichloromethyl,difluoromethyl, trifluoromethyl (CF₃), perfluoroethyl,2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

As used herein, the term “amino” means —NH₂. The term “alkylamino” meansa nitrogen moiety having one straight or branched unsaturated aliphatic,cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g.,—NH(alkyl). The term “dialkylamino” means a nitrogen moiety having attwo straight or branched unsaturated aliphatic, cyclyl, or heterocyclylradicals attached to the nitrogen, e.g., —N(alkyl)(alkyl). The term“alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and“heterocyclylamino.” The term “arylamino” means a nitrogen moiety havingat least one aryl radical attached to the nitrogen. For example,—NHaryl, and —N(aryl)₂. The term “heteroarylamino” means a nitrogenmoiety having at least one heteroaryl radical attached to the nitrogen.For example —NHheteroaryl, and —N(heteroaryl)₂. Optionally, twosubstituents together with the nitrogen can also form a ring. Unlessindicated otherwise, the compounds described herein containing aminomoieties can include protected derivatives thereof. Suitable protectinggroups for amino moieties include acetyl, tertbutoxycarbonyl,benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but isnot limited to, NH(C₁-C₁₀alkyl), such as —NHCH₃, —NHCH₂CH₃,—NHCH₂CH₂CH₃, and —NHCH(CH₃)₂. Exemplary dialkylamino includes, but isnot limited to, —N(C₁-C₁₀alkyl)₂, such as N(CH₃)₂, —N(CH₂CH₃)₂,—N(CH₂CH₂CH₃)₂, and —N(CH(CH₃)₂)₂.

The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as definedabove, except where one or more substituted or unsubstituted nitrogenatoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl,or alkynyl. For example, an (C₂-C₆) aminoalkyl refers to a chaincomprising between 2 and 6 carbons and one or more nitrogen atomspositioned between the carbon atoms.

The terms “hydroxy” and “hydroxyl” mean the radical —OH.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto, and can berepresented by one of —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined herein. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl. Exemplary alkoxy groups include, but are notlimited to O-methyl, O-ethyl, O-n-propyl, O-isopropyl, O-n-butyl,O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O-hexyl, O-cyclopropyl,O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.

As used herein, the term “carbonyl” means the radical —C(O)—. It isnoted that the carbonyl radical can be further substituted with avariety of substituents to form different carbonyl groups includingacids, acid halides, amides, esters, ketones, and the like.

The term “carboxy” means the radical —C(O)O—. It is noted that compoundsdescribed herein containing carboxy moieties can include protectedderivatives thereof, i.e., where the oxygen is substituted with aprotecting group. Suitable protecting groups for carboxy moietiesinclude benzyl, tert-butyl, and the like. As used herein, a carboxygroup includes —COOH, i.e., carboxyl group.

The term “ester” refers to a chemical moiety with formula —C(═O)OR,where R is selected from the group consisting of alkyl, cycloalkyl,aryl, heteroaryl and heterocycloalkyl.

The term “cyano” means the radical —CN.

The term “nitro” means the radical —NO₂.

The term, “heteroatom” refers to an atom that is not a carbon atom.Particular examples of heteroatoms include, but are not limited tonitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes amoiety where the atom by which the moiety is attached is not a carbon.Examples of heteroatom moieties include —N═, —NR^(N)—, —N⁺(O⁻)═, —O—,—S— or —S(O)₂—, —OS(O)₂—, and —SS—, wherein R^(N) is H or a furthersubstituent.

The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, asdefined above, where the oxygen atom is replaced with a sulfur. Inpreferred embodiments, the “alkylthio” moiety is represented by one of—S-alkyl, —S-alkenyl, and —S-alkynyl. Representative alkylthio groupsinclude methylthio, ethylthio, and the like. The term “alkylthio” alsoencompasses cycloalkyl groups, alkene and cycloalkene groups, and alkynegroups. “Arylthio” refers to aryl or heteroaryl groups.

The term “sulfinyl” means the radical —SO—. It is noted that thesulfinyl radical can be further substituted with a variety ofsubstituents to form different sulfinyl groups including sulfinic acids,sulfinamides, sulfinyl esters, sulfoxides, and the like.

The term “sulfonyl” means the radical —SO₂—. It is noted that thesulfonyl radical can be further substituted with a variety ofsubstituents to form different sulfonyl groups including sulfonic acids(—SO₃H), sulfonamides, sulfonate esters, sulfones, and the like.

The term “thiocarbonyl” means the radical —C(S)—. It is noted that thethiocarbonyl radical can be further substituted with a variety ofsubstituents to form different thiocarbonyl groups including thioacids,thioamides, thioesters, thioketones, and the like.

“Acyl” refers to an alkyl-CO— group, wherein alkyl is as previouslydescribed. Exemplary acyl groups comprise alkyl of 1 to about 30 carbonatoms. Exemplary acyl groups also include acetyl, propanoyl,2-methylpropanoyl, butanoyl and palmitoyl.

“Aroyl” means an aryl-CO— group, wherein aryl is as previouslydescribed. Exemplary aroyl groups include benzoyl and 1- and2-naphthoyl.

“Arylthio” refers to an aryl-S— group, wherein the aryl group is aspreviously described. Exemplary arylthio groups include phenylthio andnaphthylthio.

“Aralkyl” refers to an aryl-alkyl- group, wherein aryl and alkyl are aspreviously described. Exemplary aralkyl groups include benzyl,phenylethyl and naphthylmethyl.

“Aralkyloxy” refers to an aralkyl-O— group, wherein the aralkyl group isas previously described. An exemplary aralkyloxy group is benzyloxy.

“Aralkylthio” refers to an aralkyl-S— group, wherein the aralkyl groupis as previously described. An exemplary aralkylthio group isbenzylthio.

“Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and t-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an H₂N—CO— group.

“Alkylcarbamoyl” refers to a R′RN—CO— group, wherein one of R and R′ ishydrogen and the other of R and R′ is alkyl as previously described.

“Dialkylcarbamoyl” refers to R′RN—CO— group, wherein each of R and R′ isindependently alkyl as previously described.

“Acyloxy” refers to an acyl-O— group, wherein acyl is as previouslydescribed. “Acylamino” refers to an acyl-NH— group, wherein acyl is aspreviously described. “Aroylamino” refers to an aroyl-NH— group, whereinaroyl is as previously described.

The term “optionally substituted” means that the specified group ormoiety is unsubstituted or is substituted with one or more (typically 1,2, 3, 4, 5 or 6 substituents) independently selected from the group ofsubstituents listed below in the definition for “substituents” orotherwise specified. The term “substituents” refers to a group“substituted” on a substituted group at any atom of the substitutedgroup. Suitable substituents include, without limitation, halogen,hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl,alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy,aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl,alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl,alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl,acyloxy, cyano or ureido. In some cases, two substituents, together withthe carbons to which they are attached to can form a ring.

For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl oraryl is optionally substituted with 1, 2, 3, 4 or 5 groups selected fromOH, CN, SH, SO₂NH₂, SO₂(C₁-C₄)alkyl, SO₂NH(C₁-C₄)alkyl, halogen,carbonyl, thiol, cyano, NH₂, NH(C₁-C₄)alkyl, N[(C₁-C₄)alkyl]₂, C(O)NH₂,COOH, COOMe, acetyl, (C₁-C₈)alkyl, O(C₁-C₈)alkyl, O(C₁-C₈)haloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, haloalkyl, thioalkyl, cyanomethylene,alkylaminyl, aryl, heteroaryl, substituted aryl, NH₂—C(O)-alkylene,NH(Me)-C(O)-alkylene, CH₂—C(O)— alkyl, C(O)— alkyl, alkylcarbonylaminyl,CH₂—[CH(OH)]_(m)—(CH₂)_(p)—OH, CH₂—[CH(OH)]_(m)—(CH₂)_(p)—NH₂ orCH₂-aryl-alkoxy; or wherein any alkyl, cycloalkyl or heterocyclyl isoptionally substituted with oxo; “m” and “p” are independently 1, 2, 3,4, 5 or 6.

In some embodiments, an optionally substituted group is substituted with1 substituent. In some other embodiments, an optionally substitutedgroup is substituted with 2 independently selected substituents, whichcan be same or different. In some other embodiments, an optionallysubstituted group is substituted with 3 independently selectedsubstituents, which can be same, different or any combination of sameand different. In still some other embodiments, an optionallysubstituted group is substituted with 4 independently selectedsubstituents, which can be same, different or any combination of sameand different. In yet some other embodiments, an optionally substitutedgroup is substituted with 5 independently selected substituents, whichcan be same, different or any combination of same and different.

An “isocyanato” group refers to a NCO group.

A “thiocyanato” group refers to a CNS group.

An “isothiocyanato” group refers to a NCS group.

“Alkoyloxy” refers to a RC(═O)O— group.

“Alkoyl” refers to a RC(═O)— group.

It should be understood that this disclosure is not limited to theparticular methodology, protocols, and reagents, etc., provided hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present disclosure, which is defined solely by the claims.The invention is further illustrated by the following example, whichshould not be construed as further limiting.

EXAMPLES Example 1: New Inhibitors for the KEAP1-Nrf2 Protein-ProteinInteraction

On average, an approved drug today costs $2-3 billion and takes over tenyears to develop¹. In part, this is due to expensive and time-consumingwet-lab experiments, poor initial hit compounds, and the high attritionrates in the (pre-)clinical phases. Structure-based virtual screening(SBVS) has the potential to mitigate these problems. With SBVS, thequality of the hits improves with the number of compounds screened².However, despite the fact that large compound databases exist, theability to carry out large-scale SBVSs on computer clusters in anaccessible, efficient, and flexible manner has remained elusive. Here wedesigned VirtualFlow, a highly automated and versatile open-sourceplatform with perfect scaling behaviour that is able to prepare andefficiently screen ultra-large ligand libraries of compounds.VirtualFlow is able to use a variety of the most powerful dockingprograms. Using VirtualFlow, we have prepared the largest and freelyavailable ready-to-dock ligand library available, with over 1.4 billioncommercially available molecules. To demonstrate the power ofVirtualFlow, we screened over 1 billion compounds and discovered a smallmolecule inhibitor (iKeap1) that engages KEAP1 with nanomolar affinity(K_(d)=114 nM) and disrupts the interaction between KEAP1 and thetranscription factor NRF2. We also identified a set of structurallydiverse molecules that bind to KEAP1 with submicromolar affinity. Thisillustrates the potential of VirtualFlow to access vast regions of thechemical space and identify binders with high affinity for targetproteins.

Repeated optimization of lead compounds and late-stage failure of drugcandidates are the primary causes of longer development times andincreased costs in drug development. Improving the quality of theinitial lead compounds would minimize these lead optimization cycles andresult in drug candidates entering (pre-)clinical phases with greaterspecificity and higher affinity. Virtual screening to identify moleculesthat bind to a specified site on a receptor protein has become animportant part of the drug discovery pipeline²⁻⁵.

Current virtual screening paradigms routinely sample only a tinyfraction, on the order of 10⁶-10⁷ molecules, of the total chemical spaceof small organic compounds suitable for drug discovery, estimated toencompass more than 10⁶⁰ molecules⁶.

However, the scale of a virtual screen is of central importance becausethe more compounds that are screened, (a) the lower the rate of falsepositives, and (b) the more favourable the quality of the lead compounds(e.g. higher-affinity binders). It was recently shown experimentallythat ultra-large scale screening improves the rate of true positives².Here we derived a probabilistic model of the true-positive rate as afunction of the number of compounds screened, and analysis of ourultra-large screen confirms that the docking score of thehighest-scoring compounds improve with the scale. Increasing the scaleof a virtual screen can improve the quality of initial hits in twodistinct ways: (1) by identifying hits with tighter binding affinity,which can result in lowered dosages and fewer off-target effects, and(2) by discovering compounds with more favourable pharmacokinetic and/orless inherent cytotoxic properties.

To increase the number of compounds evaluated in a virtual screen byorders of magnitude and make it accessible to any researcher, there is adire need for a platform that can integrate all the tasks in the virtualscreening process. Such a platform should ideally (1) scale linearlywith the number of CPUs, (2) efficiently handle billions of files, (3)minimize input and output (I/O) load, (4) run robustly (e.g. skipincorrectly encoded ligands, resist temporary I/O problems, and resumefollowing unexpected termination), (5) run on any type of computingcluster (including cloud platforms), and (6) be user-friendly and easyto use for non-computational scientists. Furthermore, to provideflexibility, a SBVS platform should be able to interface with a varietyof docking programs, support both rigid and flexible receptor docking,test multiple docking scenarios in a single workflow, allow forconsensus and ensemble docking, and carry out multiple replicas of thesame docking scenario. Lastly, to democratize access, facilitatewidespread usage, and catalyse further development, such a platformwould need to be open source.

With these requirements in mind, we designed VirtualFlow, an open-sourceplatform that is able to screen chemical space on an unprecedentedscale. Screening one billion compounds on a single processor core, withan average docking time of 15 seconds per ligand, would take 475 years.By contrast, VirtualFlow can dock one billion compounds in approximatelytwo weeks by leveraging 10,000 CPU cores simultaneously. Such highperformance computing facilities are available to researchers throughseveral potential sources, including local institute computer clusters,national super-computing centres, or cloud computing platforms.

Targeting KEAP1 Using VirtualFlow

To test the advantages of ultra-large-scale in silico screening and theperformance of the VirtualFlow platform we decided to target thechallenging and therapeutically relevant protein-protein interaction(PPI) between nuclear factor erythroid-derived 2-related factor 2 (NRF2)and Kelch-like ECH-associated protein 1 (KEAP1). NRF2 is a masterregulator of cellular resistance to oxidative stress and cellularrepair⁷. Under unstressed conditions, NRF2 is sequestered by KEAP1, anE3 ubiquitin ligase substrate adaptor, and targeted for degradation⁸.However, upon oxidative stress, reactive oxidants dissociate NRF2 fromKEAP1 and NRF2 translocates to the nucleus to activate itstranscriptional program of approximately 250 genes⁹. The NRF2-KEAP1pathway is critical in protecting the cell under oxidative stress andinflammation and is implicated in a number of diseases¹⁰. There are tendrugs targeting KEAP1 that are in clinical trials and nine more that areat the preclinical stage¹⁰. Using VirtualFlow, we screened ˜1.3 billioncompounds (˜1 billion compounds from the Enamine REAL Library and ˜330million compounds from the ZINC library) against the NRF2 interactioninterface on KEAP1. First, we would like to describe the salientfeatures of VirtualFlow and its scalability.

Characteristic Features of VirtualFlow

One of the key features of VirtualFlow is its linear scaling behaviour(O(N)) with respect to the number of CPUs and nodes utilized.VirtualFlow can run on computer clusters operated with any of the majorresource managers (SLURM¹¹, Moab/TORQUE¹², PBS¹³, LSF¹⁴ and SGE¹⁵), andcompatibility with additional job schedulers can be easily added. ThusVirtualFlow is also ideally configured for cloud computing platformslike Amazon's Web Services (AWS), Microsoft's Azure and Google's CloudPlatform (GCP). VirtualFlow is able to run autonomously from the firstto the last ligand in the screening pipeline, a feature facilitated byautomatic submission of new batch system jobs. The workflow can bemonitored and controlled in real time. The VirtualFlow package consistsof two applications that work seamlessly together: The VFLP (VirtuaFlowfor Ligand Preparation) module, which prepares small molecules forscreening; and the VFVS (VirtualFlow for Virtual Screening) module,which executes the virtual screening procedures. The separation ofligand preparation and virtual screening is desirable because the sameready-to-dock ligand library can be used in any number of VFVS virtualscreens.

VirtualFlow for Ligand Preparation (VFLP)

VFLP (VirtualFlow for Ligand Preparation) prepares ligand databases byconverting them from the SMILES format into any desired target format(e.g. the PDBQT format, which is required by many of the AutoDock-baseddocking programs). VFLP uses ChemAxon's JChem package as well as OpenBabel to desalt ligands, neutralize them, generate (even multiple)tautomeric states, compute protonation states at specific pH values,calculate 3D coordinates, and convert the molecules into desired targetformats.

Preparation of the Enamine REAL Library

Commercially available compounds constitute the most interesting subsetof the chemical space, since these compounds can be readily purchased.The largest vendor library available today is the REAL library ofEnamine, containing approximately 1.4 billion make-on-demand compounds(as of October 2019 the ZINC 15 database contained 1.46 billioncompounds, but only provided 630 million molecules in a ready-to-dockformat). We have used VFLP to convert the ˜1.4 billion compounds of theREAL library into PDBQT format (see Methods), and have made it freelyavailable on the VirtualFlow homepage, accessible via a graphicalinterface (FIGS. 3A-3D). The entire database has a six-dimensionallattice architecture, the general concept of which was modelled afterthe ZINC 15 database¹⁶, where each dimension corresponds to aphysico-chemical property of the compounds (molecular weight, partitioncoefficient, number of hydrogen bond donors and acceptors, number ofrotatable bonds, and the topological polar surface area). Thepreparation of ligands using VFLP is a one-time effort.

VirtualFlow for Virtual Screening (VFVS)

To set up a virtual screen with VFVS, a set of docking scenarios isspecified by the user. Docking scenarios are defined by the choice ofthe external docking program, the receptor structure, and the dockingparameters (which include the pre-defined docking surface on thereceptor, residues on the receptor that are allowed to be flexibleduring docking, and the rigor of the docking routine). VirtualFlowcurrently supports the following docking programs: AutoDock Vina¹⁷,QuickVina 2^([18]), Smina (which includes the Vinardo and AutoDock 4scoring functions)¹⁹, AutoDockFR (ADFR)²⁰, QuickVina-W⁵, VinaXB²¹, andVinaCarb²². By supporting an array of different docking programs, VFVScan be used in a variety of cases by leveraging the unique advantages ofeach program. VFVS allows the specification of multiple dockingscenarios to be carried out for each ligand, enabling consensus dockingprocedures, as well as ensemble docking procedures^(23,24). VirtualFlowis also amenable to the integration of other docking programs that arenot currently a part of this platform.

Scaling Behaviour of VFVS

In order to measure the scaling behaviour of VFVS, we measured theperformance on two local clusters, LC1 and LC2. On LC1, we used 18,000CPU cores of heterogeneous composition (different models of Intel Xeonand AMD Opteron processors), whereas on LC2 we employed up to 30,000Intel Xeon 8268 CPUs. The scaling behaviour was effectively linear inboth cases (i.e., O(N), where N is the number of cores). These resultsmeet theoretical expectations since there is no direct communicationbetween the processes running in parallel, which is key to perfectscaling behaviour without bounds. The independence of its parallelprocesses means that VirtualFlow is expected to scale linearly even ifmillions of cores are used. We also tested the performance of theplatform on cloud-based computing systems including GCP and AWS. On theGCP we carried out large-scale benchmarks with up to 160,000 CPUs, anddespite this massive scaling in CPU volume, VirtualFlow still exhibitedlinear scaling. A typical high-throughput screen, such as the onedescribed in this study, of 1 billion compounds will take ˜15 hours onthe GCP with 160,000 CPUs, making VirtualFlow suitable for the highlyanticipated exascale computing age.

Multistaged Virtual Screens with VFVS

VFVS can also be used to organize virtual screens with multiple stagesto substantially increase the quality of the results. In themulti-staging approach, several virtual screens are executed insuccession. The number of top-scoring compounds that advance from onestage to the next is successively reduced, with concomitant increases indocking accuracy and computational cost.

Using VFVS to Screen 1.3 Billion Ligands

In order to validate the performance of VFVS we screened a virtuallibrary of 1.3 billion commercially available compounds (˜330 millioncompounds from the ZINC 15 database¹⁶, and ˜1 billion compounds from theEnamine REAL library) against KEAP1. % It should be noted that there issome overlap of compounds between the two libraries.

This effort was completed in around four weeks, using on averageapproximately 8,000 cores on a heterogeneous Linux cluster.

To illustrate the benefit of an ultra-large-scale screen, we chose asubsets of the ligands (0.1, 1, 10, and 100 million compounds) randomlyfrom the ˜1 billion compound screen of the REAL library and consideredthe scores of the top 50 compounds. As the scale of the screenincreased, the average docking score increased thus improving thechances of identifying tighter binders. This in turn leads to highertrue hit rates and tighter experimental binding affinities, as predictedby a probabilistic model experimentally demonstrated previously².

To demonstrate VirtualFlow in a multi-staging context we subjected thetop ˜3 million ranking compounds from the primary virtual screen to arescoring procedure. In stage-2, the 13 residues of KEAP1 at the NFR2interaction interface were allowed to be flexible. This flexibilityaccounted for the movement/dynamics of the amino acids at the bindinginterface, not captured by a static structure. In the re-scoringprocedure we utilized two different docking programs (Smina Vinardo andAutoDock Vina), and two replicas of each docking scenario were carriedout to further increase the conformational space sampled during thedocking runs. The necessity of multi-stage screening depends on thetarget of choice and the computational resources available, but thistype of virtual screen is particularly useful in cases where dynamics atthe docking interface is expected to play a significant role.

Experimental Validation

From the in silico screen described above, we chose 590 hits forexperimental validation. Of these, 492 compounds were from the top 0.03%of stage-2 screen and 98 compounds were from the top 0.0001% of stage-1.Hits from stage-1 were ordered to compare the true hit rate betweenstage-1 and stage-2 hits, in a multistage setting. In addition to theranking by docking score, the choice of these compounds was based onfactors like drug-likeness, availability for procurement, ligandefficiency and chemical diversity. We used four established biophysicalmethods: fluorescence polarization (FP), surface plasmon resonance(SPR), nuclear magnetic resonance (NMR), and bio-layer interferometry(BLI) to experimentally validate the binding of the VirtualFlow-derivedhits to KEAP1. FP and SPR were initially used in a high-throughputfashion (Level-1) to detect binding and the compounds identified herewere subsequently validated with more scrutiny in a detailed andlow-throughput fashion (Level-2). We used a recombinantly expressed andpurified Kelch domain of mouse KEAP1, henceforth referred to as KEAP1. Adetailed description of the experimental procedure is provided in themethods section. Of these four biophysical methods, FP and BLI detectthe ability of the hits to displace the NRF2 peptide from KEAP1,identifying hits we refer to as displacers. SPR and NMR directly detectbinding of hits to KEAP1, identifying hits referred to as binders.VirtualFlow identifies molecules that potentially bind to theNRF2-interacting interface on KEAP1, but the in silico screen isperformed using KEAP1 alone, in the absence of NRF2. The NRF2 bindingsurface on KEAP1 is part of the deep pocket/tunnel of the KEAP1 β-barrelwith NRF2 binding to the entrance of this tunnel. However, somecompounds could bind more tightly by inserting deep into this centraltunnel of KEAP1 rather than embracing the surface like the NRF2 peptide,and/or bind to parts of KEAP1 not engaged by NRF2. Such binders mightnot effectively disrupt the interaction with NRF2, while still engagingKEAP1 with high affinity (FIG. 6 ). In our experimental validation weidentified both displacers and binders.

Out of the cherry-picked 590 compounds, 69 were confirmed to bind toKEAP1 by Level-2 SPR. To assess the ability of the compounds to displacethe NRF2 peptide we used the FP assay. Ten compounds were confirmed tobe displacers with an IC₅₀<60 μM by FP and all of them were identifiedas a binder by Level-2 SPR. Interference by autofluorescence from thecompounds themselves prevented the analysis of some of the compounds byFP. Thus, we used BLI as an orthogonal assay to assess the ability ofthe compounds to displace NRF2. The binding affinity of the NRF2 peptideto KEAP1 as measured by BLI was 1.86 nM which is similar to thatmeasured by FP, 3.67 nM (Extended Data FIGS. 2A and 2B). 40 compounds ofthe 69 SPR Level-2 active compounds were able to disrupt the NRF2-KEAP1interaction as observed by BLI. Of these 40 compounds, 16 were able todisplace NRF2 from KEAP1 at a compound concentration of 20 μM, while all40 compounds could do so at 100 μM. Using BLI, we were able to identifydisplacers that were missed by FP due to autofluorescence (an example isshown in Extended Data FIGS. 5A-5F). We tested all the SPR Level-2active compounds for potential aggregation by Dynamic Light Scattering(DLS). We identified seven compounds that aggregated in the DLS assayand hence were not considered for further evaluation (Table 6). Based onthe SPR Level-2 and the FP Level-2 binding data, we selected 23compounds for SPR Level-3 experiments to determine the binding affinity.All 23 compounds had affinities in the low micromolar to nanomolarrange, and 12 compounds had submicromolar K_(d) values. From these 23compounds, we tested the binding of six compounds (iKeap1, 2, 7, 8, 9and 22) to KEAP1 by a suite of NMR-based ligand-detected experiments.Out of these six compounds, five are displacers and one of them (iKeap9)is a binder. These six compounds were selected on the basis of thesolubility constraints of the NMR experiments, the SPR K_(d) value,and/or their ability to displace the peptide. We used differential linebroadening (DLB), saturation transfer difference (STD),Car-Purcell-Meiboom-Gill (CPMG)-based transverse relaxation timeexperiments, and protein-observed ¹H—¹³C heteronuclear multiple-quantumcorrelation (HMQC) experiments to confirm binding of the compounds toKEAP1. The ligand-detected NMR experiments confirmed that all six of thetested SPR Level-3 active compounds bind to KEAP1 (FIGS. 1A-1H, 4A-4Hand 5A-5F). Protein-detected ¹H—¹³C HMQC experiments show that thecompounds engage KEAP1 in a specific manner, at the targeted NRF2binding site. In the absence of resonance assignments, we use the factthat the compounds perturb a subset of KEAP1 resonances affected by theaddition of the NRF2 peptide as evidence for competitive binding. Thesecompounds are shown in FIGS. 7A-9D. Details about the other activecompounds is provided in Supplementary Information Section B.

Two of our top hits, iKeap1 and iKeap2 are able to displace the NFR2peptide from KEAP1. Both of the compounds are predicted to engage theNRF2 binding pocket on KEAP1, located at the entrance to the tunnelformed by the β-barrel. (FIGS. 1A and 1B). In comparison to iKeap2,iKeap1 descends deeper into this central tunnel of KEAP1. SPR resultsshowed that iKeap1 and iKeap2 bind to KEAP1 with a binding affinity of114 nM and 158 nM, respectively (FIGS. 1C and 1D). NMR-basedligand-detected experiments confirmed that both iKeap1 and iKeap2directly bind to KEAP1 (FIGS. 1E and 1F). FP assays showed that iKeap1is able to displace NRF2 peptide with an IC₅₀ of 258 nM and iKeap2displaces the NRF2 peptide with an IC₅₀ of 2.7 μM (FIGS. 1G and 1H). BLImeasurements additionally confirmed that both iKeap1 and iKeap2 are ableto displace the NRF2 peptide from KEAP1. iKeap1 exhibits similarity to apreviously reported naphthalene-based compound with a lower IC₅₀(IC₅₀=2.7 μM; compound C17 in Table 2 and FIGS. 3A-3D)²⁵. C17 wasidentified as the best hit in a high-throughput screen (HTS) of 270,000compounds²⁵.

We would also like to highlight iKeap7, which has the highest affinityas assayed by SPR (K_(d)=15 nM) and displaces the NRF2 peptide with anIC₅₀ of 38.2 μM (FIGS. 5A-5F). It should be noted that of the 14 hitsdescribed in the manuscript, only two hits, namely iKeap2 and iKeap7,contain pan-assay interference compound (PAINS) sub-structures. However,we performed a series of orthogonal binding assays, which confirmed thatiKeap2 and iKeap7 are not experimental false positives. For details anddiscussion on how we verified that our experimental results were notaffected by PAINS see Supplementary Section B.

Typically PPIs have a larger interaction interface as compared to thatof the active site of an enzyme. Hence the in silico screen can identifybinders that either partially overlap with the binding site of theinteracting protein, such as iKeap9 (FIGS. 4A-4H), or those that bind ina manner which energetically favours the formation of theprotein-protein complex. Examples of the latter, referred to as glues,have been previously described in the literature²⁶.

An Open Source Platform

To allow VirtualFlow to be used widely and develop dynamically, it isset up as a free and open source (FOSS) project. GPU support is plannedfor the future and will be incorporated into VirtualFlow both nativelyand via external docking programs such as Gnina²⁷. We encouragescientists to join the project and contribute to improving existingfeatures, adding new features and functionality. The primary homepage ofVirtualFlow, which provides additional resources, can be accessed athttps.//www.virtual-flow.org.

Outlook

VFVS can be used to search extremely large regions of the chemicalspace, which is the key to identifying promising small-molecule binders.VFVS is able to accomplish this by efficiently utilizinghigh-performance computing resources, which will continue to increase inavailability and power in the years to come, and novel virtual screeningdatabases such as the Chemical Universe Databases (GDBs), which containbillions to trillions of compounds, are still waiting to be explored²⁸.

Methods

Parallelization of the Virtual Screen with VirtualFlow

VirtualFlow employs four levels of parallelization in a hierarchicalmanner to permit it to run on batch system-managed Linux clusters of anyconfiguration while allowing for perfect scaling behaviour. Eachinstance of VirtualFlow can submit multiple jobs, each job may useseveral job steps (currently only supported when using SLURM andMoab/TORQUE/PBS as the resource manager, while for SGE and LSF onlysingle job steps per job are possible), one job step is able to executean arbitrary number of queues, and each queue executes the externalprograms which are processing the ligands. These programs may beadditionally parallelized internally, for instance via multithreading.

Workload Balancing

When processing ligands in parallel, there needs to be a mechanism whichmakes sure that each ligand is treated only once. However, one mainproblem with parallelization is that most cluster file systems are tooslow to work off a single simple task list. This is because whendifferent processes access the file at the same time, clashes can occur,as it may take up to several seconds until one job sees the changes madeto a file by another job. These latency problems also mean that filelocking mechanisms do not prevent these clashes. The standard solutionfor solving this kind of problem is to let different processescommunicate directly with each other or via a central master process.However, in most cases, this results in sub-linear scaling behaviour,which normally worsens as more and more parallel running processesbecome involved. Moreover, many advanced parallelization methods such asMPI or OpenMP, do not allow for inter-job communication, while in manycases multiple simultaneously running jobs are needed. Therefore, inorder to maintain perfect scaling behaviour which allows multiple jobsand a virtually unrestricted number of CPUs, we have developed anadvanced task-list mechanism. The key is to minimize the number ofinstances that the parallel processes need to access the task list. Themechanism we have implemented requires only a single access per batchsystem job, each of which can contain a large number of parallel runningprocesses. For this purpose, we have implemented a workload balancer,which distributes the tasks from the central task list at the beginningof each job to all the queues belonging to it. The central task listcontains collections of ligands as elementary components (rather thanindividual ligands), and the workload balancer takes into account thelength of each collection when distributing them among the queues. Thisapproach dramatically reduces the number of times the central task listhas to be accessed. For example, if the workflow employs 10 jobs inparallel, and each job runs on 100 nodes with a wall time (real runtime) of one week and 24 CPU cores per node, and one ligand requiresapproximately 30 seconds to be docked, then the central task list needsto be accessed only 10 times per week to feed a total of 24,000 parallelrunning queues (assuming each queue runs on one CPU core). In this case,approximately 483,840,000 ligands are processed in one week, which meansthat the advanced task list approach reduced the number of accesses tothe central task list by a factor of 48,384,000 in comparison to thenumber needed by a trivial task list approach (one access per ligandprocessed). This factor can be improved even further depending on thejob size and the cluster wall time. In case two parallel processes wantto access the central task list simultaneously, two backup mechanismswere implemented. The first mechanism is a time-dispersion mechanism,which spreads out simultaneously arriving jobs in time, and furtherstalls subsequent jobs until the workload balancer of the current job isfinished. If this mechanism should fail to prevent a simultaneous accessevent, which could result in a damaged or empty task file, a secondmechanism restores the task list using an automatically backed-up copyof a previous version of the central task list.

Reduction of I/O Load

One of the potential bottlenecks of computer clusters is the I/O loadthey can handle, even when they utilize shared cluster file systems withhigh bandwidth. The limit of the I/O capacities of a cluster can beeasily reached if many small processes that individually handle theirI/O and use the shared file system are running in parallel. Thiscircumstance can pose a serious problem when running large-scaleworkflows with thousands of queues working in parallel, and can easilylead to crashing the cluster file system. To address this problem anddramatically minimize the load on the shared file system, VirtualFlow isable to perform most I/O operations on the local temporary file systemsof the computing nodes, which are normally fast RAM-based (virtual)drives readily available on any Linux system (usually/dev/shm). Thefinal output files are then stored in batches at large time intervals onthe permanent cluster file system.

Preparation of the Ligand Databases

One of the ligand databases which was screened originates from the stateof the ZINC 15 database in the autumn of 2016. Approximately 330 millioncompounds were downloaded in the SMILES format and converted intothree-dimensional PDBQT files with VFLP because, at the time, the ZINC15 database only provided a fraction of the compounds in a ready-to-dockformat. During the conversion, the molecules were protonated withChemAxon's cxcalc and the three-dimensional structure of the ligand wascomputed by ChemAxon's molconvert tool²⁹. If protonation or thegeneration of the three-dimensional structure failed, Open Babel³⁰ wasemployed as a fallback option. Other preparation steps, such asdesalting, were not carried out on these compounds as they had alreadyundergone these basic preparation steps for the ZINC 15 database.

We also prepared the compounds in the REAL database provided byEnamine³¹. Approximately 700 million partially-stereospecific SMILESwere expanded into fully stereospecific SMILES, resulting in around 1.4billion molecules. These were then prepared with VFLP into aready-to-dock format. Specifically, the compounds were desalted andneutralized with ChemAxon's cxcalc, major tautomers were computed withcxcalc, and then protonated with cxcalc (using Open Babel as fallback),the 3D coordinates were computed with ChemAxon's molconvert²⁹ (usingOpen Babel as a fallback), and finally converted into the PDBQT formatwith Open Babel. This library has been made available via an interactiveweb interface (Supplementary Section C). The scaling behaviour of VFLPwas measured on the GCP up to 20,000 CPU cores (data not shown).

Computation Time of VFVS

The total computation time (T) is directly proportional to the number ofligands screened (N) and the processing time per ligand (P), andinversely proportional to the number of CPUs used (C):

T≈(P*N)/C

The processing time per ligand (P) depends mainly on the specificdocking scenario (which includes the receptor and all the possibledocking options/parameters) and the speed of CPUs used, and can beapproximated by the equation

P≈(E*Θ+ζ)/η

where η is a factor representing the CPU speed relative to a referenceCPU, E is the docking exhaustiveness parameter (elaborated in the nextparagraph below), Θ is the docking time per unit exhaustiveness on thereference CPU, and ζ is the initial setup time required by the dockingprogram on the reference CPU. For a typical case of a large-scalefirst-stage virtual screen on one of the newer Intel CPUs, the averageprocessing time per ligand (P) is roughly 5 seconds using the fastestdocking settings. It follows that when 5,000 CPUs are used, the totalscreening time for 100 million compounds will be roughly 30 hours.

Relationship Between the Exhaustiveness Parameter and the Docking Time

The time to dock a single molecule depends on the number ofconformations that are sampled, and this number is largely independentof the size of the docking box or surface area. The number ofconformations sampled can be controlled by the exhaustiveness parameterof the docking programs. Docking time has a linear dependency on theexhaustiveness parameter. The inset in the graph shows the slope foreach of the docking programs, providing an estimate of the degree ofdependency between the computational time and the exhaustivenessparameter for individual docking programs.

Since most docking programs utilize a probabilistic search algorithm,the results of separate iterations with the same starting set-up candiffer. This circumstance can be beneficial as it can be more efficientto carry out multiple less exhaustive docking iterations than to run onehighly exhaustive iteration. The exhaustiveness here is a measure of theextent to which the conformational space of the ligand, and potentiallythe protein side chains, is explored by the search algorithm during thedocking procedure. In light of this, VFVS can be configured to carry outmultiple replicas per docking scenario, thus improving the overallefficiency.

Lead Optimization Using VFVS

The operational flexibility enables VFVS to also be used during lead. Inthis context, a library of analogues of a chosen lead compound can beprepared with VFLP and screened by VFVS with high docking accuracy (e.g.setting the exhaustive parameter to a high value, allowing specificamino acids in the binding interface to be flexible, using multipledocking programs, and/or multiple receptor (backbone) conformations),which can considerably accelerate the lead optimization process.

Parameters of the Virtual Screen Against the KEAP1 Target

For the virtual screening validation test, the crystal structure of theKEAP1 Kelch domain (PDB-ID 5FNQ⁹) was used. The protein was stripped ofall small molecules present (including water), was protonated atphysiological pH, and then converted into PDBQT format usingAutoDockTools³².

The NRF2 binding interface on KEAP1 was chosen as the target of thescreening, and the exact location determined by previously publishedco-crystal structures of KEAP1 and the NRF2 peptide (such as PDB ID:4IFL). The in silico screen was carried out as follows: VFVS used thedocking program QuickVina 2 in an initial (primary) virtual screen withthe mouse KEAP1 as a rigid receptor structure.

In this primary virtual screening, the docking search space was arectangular parallelepiped (i.e. a cuboid) of size 15.0×16.5×14.275 Å.The exhaustiveness parameter was set to 1, which favours fastcomputational times. The quality of individual docking results, andtherefore the ranking, depends largely on the external docking programchosen (which is independent of VirtualFlow).

In the rescoring procedure, the following amino acid side chains at thebinding interface were allowed to be flexible: Tyr334, Arg380, Asn382,Arg415, Cys434, His436, Ile461, Phe478, Arg483, Ser508, Tyr525, Tyr572and Phe577. AutoDockTools was used to generate the rigid and flexiblereceptor structures in PDBQT format. The exhaustiveness was set to 1,and two replicas (iterations) were carried out of each docking scenario(with Smina Vinardo and AutoDock Vina as the docking programs). The sizeof the docking box was set to 27.0×27.0×24.0 Å.

Expression and Purification of GST-KEAP1:

A codon optimized vector of the mouse KEAP1 Kelch domain (residues322-624) cloned into a pGEX-6P-3 vector with BamHI and XhoI cloningsites, and an NRF2 peptide (AFFAQLQLDEETGEFL (SEQ ID NO: 1) with anN-terminal tetramethylrhodamine (TAMRA) fluorophore were purchased fromGenScript USA Inc. (NJ, USA). The pGEX-6P-3 vector contains anN-terminal glutathione S-transferase (GST) tag which is expressed as afusion with the target sequence, resulting in a gene product that willhenceforth be referred to as GST-KEAP1. The vector carrying GST-KEAP1was transformed into BL21(DE3) E. coli. The transformed cells were grownat 37° C. to an optical density of 0.6 at a measurement wavelength of600 nm and protein expression was induced with 0.5 mM isopropylβ-D-1-thiogalactopyranoside (IPTG). The cells were allowed to grow for12-16 h at 18° C. and subsequently harvested by centrifugation at 4,200rpm for 20 min at 4° C.

To purify GST-KEAP1, cell pellets from 2 L of culture were resuspendedin 40 mL of GST binding buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1mM EDTA) supplemented with 3.5 mM β-mercaptoethanol and proteaseinhibitors (Roche). Cells were lysed by sonication, the insolublefraction was removed by centrifugation at 16,000 rpm and the solublefraction was applied to 10 mL of GST slurry (GoldBio, MO). Thesuspension was mutated for 4 hours at 4° C., and the unbound fractionwas removed by gravity-flow chromatography. The slurry was washed twicewith GST binding buffer supplemented with 3.5 mM β-mercaptoethanol. Thebound fraction was eluted from the slurry with 20 mM reduced glutathionein GST binding buffer. The resulting eluate was loaded on a Superdex 200size exclusion column (SEC) pre-equilibrated in SEC buffer (20 mMTris-HCl, pH 8.0, 50 mM NaCl, 10 mM dithiothreitol).

Fluorescence Polarization (FP) Assays:

Dissociation constant of the NRF2-KEAP1 interaction: We prepared 2 nMTAMRA-NRF2 peptide in FP buffer (20 mM Tris-HCl pH: 8.0, 50 mM NaCl, 10mM DTT, 2 mM 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS), 0.005% BSA, 1% DMSO) in Corning 3575 384-well plates, toestablish the dissociation constant (K_(d)) of the TAMRA-NRF2-GST-KEAP1interaction. GST-KEAP1 was titrated into the TAMRA-NRF2 peptide startingat a concentration of 76 NM GST-KEAP1 followed by two-fold dilutions fora total of 24 points. A K_(d) of 3.67±0.35 nM was determined for theinteraction (FIGS. 2A and 2B).

FP Level-1 (high-throughput screening of compounds): All 590 compoundsordered for testing were dissolved in DMSO-d₆ to a final concentrationof 10 mM. Two AB1056 (Abgene, NH, USA) plates were prepared as sourceplates for screening. The first source plate contained 11 μL of each ofthe 10 mM compounds. The second source plate was filled with 9 NL DMSOand 1 μL from the first source plate was transferred into the second viapin transfer with a Vprep liquid handling pipetting station (Agilent,CA), resulting in a final concentration of 1 mM for each compound in thesecond source plate. 384-well Corning 3575 (Corning, N.Y., USA) assayplates were pre-loaded with 7 nM GST-KEAP1 in FP buffer (30 μL/well).300 nL, 100 nL, and 33 nL volumes were transferred from each sourceplate (the 10 mM and 1 mM plates) to pre-loaded 384-well assay plates.The assay plates were incubated for 1 hour at room temperature before 2nM TAMRA-NRF2 peptide was added to each well with an HP D300(Hewlett-Packard, CA). After 3 hours of incubation at room temperature,fluorescence polarization (excitation 485 nm/emission 520 nm) wasmeasured using an EnVision plate reader (PerkinElmer, MA, USA). Thisassay resulted in six-point titrations, which are not sufficient tocalculate accurate IC₅₀ values, but allow for the selection of topbinders.

FP Level-2 (screening of top hits): The 27 compounds which were activein the FP Level-1 assay were subjected to a second 24-point FP screen(Level-2), starting from 500 μM compound followed by 1.5-fold serialdilution. For the best compound, iKeap1, the starting concentration waslowered to 30 μM and the following concentrations were used in thetitration: 30.00 μM, 21.60 μM, 15.50 μM, 11.10 μM, 8.00 μM, 5.76 μM,4.14 μM, 2.98 μM, 2.15 μM, 1.54 μM, 1.11 μM, 0.80 μM, 0.576 μM, 0.414μM, 0.298 μM, 0.215 μM, 0.154 μM, 0.111 μM, 0.080 μM, 0.0606 μM, 0.0459μM, 0.0348 μM, 0.0264 μM, 0.02 μM. The measurements were carried out intriplicate, and the three data points for each concentration averaged.IC₅₀ values were determined by fitting the averaged data points to afour parameter logistic curve using the non-linear least squares methodprovided by the SciPy library for Python³³. The standard error (seeTable 5) on the IC₅₀ was computed by taking the square root of thediagonal of the parameter covariant matrix.

Bio-Layer Interferometry (BLI) Assays

Bio-layer interferometry binding and displacement assays: NRF2-KEAP1binding BLI experiments were performed on an Octet RED384 (ForteBio,Menlo Park, Calif., USA) using streptavidin-coated Dip and ReadBiosensors (ForteBio) and 384 well plates with 120 μL volume. Thesensors were incubated for 5 minutes in 500 nM biotinylated NRF2 peptidein binding buffer (10 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% (v/v) Tween20with 0.5 mM TCEP and 1% DMSO). To test for nonspecific binding ofGST-KEAP1 protein, reference tips were incubated in buffer alone. Thetips were washed with buffer for 2 minutes to obtain a baseline readingand then transferred to wells in various concentrations of GST-KEAP1protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.75 nM, 3.375 nM, 1.679 nM,0.844 nM) for 10 minutes. After measuring association, tips were movedto wells containing buffer, and dissociation was measured for 5 minutes.The data were processed and analysed using the Octet data analysissoftware version 11.0 (ForteBio, Inc., Menlo Park, Calif., USA). Theassociation-dissociation curve for each concentration was fitted using a1:1 model given by the equations

$\begin{matrix}{R_{t}^{on} = {\frac{k_{on} \cdot C}{{k_{on} \cdot C} + k_{on}}{R_{\max}\left( {1 - e^{{- {({{k_{on} \cdot C} + k_{off}})}} \cdot t}} \right)}}} & (3)\end{matrix}$ $\begin{matrix}{R_{t}^{off} = {R_{eq} \cdot e^{{- k_{off}} \cdot t}}} & (4)\end{matrix}$

where R_(t) ^(on) and R_(t) ^(off) are the BLI signals at time t, R_(eq)is the equilibrium response, k_(on) is the association rate constant,k_(off) is the dissociation rate constant, C is the analyte (protein)concentration, and R_(eq) is the signal level at the equilibrium ofassociation which depends on the analyte (protein) concentration and themaximal capacity (R_(max)) of the sensor surface. By computing the ratiok_(off), k_(on) the apparent equilibrium constant K_(d) is obtained. Theresulting apparent K_(d) values were averaged.

Compound screening by BLI displacement assay: The BLI displacementassays were setup as described above. The biotinylated NRF2 peptide wasused at a concentration of 500 nM and GST-KEAP1 protein was used at aconcentration of 25 nM. The compounds were used at concentrations of 20and 100 NM, and pre-incubated with GST-KEAP1 protein. The associationphase was measured in the well containing compound with GST-KEAP1protein for 10 minutes, and followed by a dissociation phase in bufferfor 5 minutes. The inhibition percentage was the average BLI signal inthe last 50 seconds of the dissociation phase, normalized against thecondition of GST-KEAP1 protein in the absence of compound. Thedose-dependent experiment with iKeap22 was carried out at 10 μM, 20 μM,40 μM, 80 μM and 100 μM compound concentration and pre-incubated with 25nM GST-KEAP1 protein.

To test for nonspecific binding of the compounds, the sensor was coupledwith biotinylated NRF2 peptide and the compounds were used at 20 μMconcentration without protein.

Surface Plasmon Resonance (SPR) Binding Assays

All SPR binding experiments were performed on a BiacoreT200 (GEHealthcare, Sweden) instrument at 25° C. in running buffer (10 mM HEPESpH 7.5, 50 mM NaCl, 0.1% (v/v) Tween20 with or without 0.5 mM TCEP, 1%DMSO). The running buffer was prepared freshly on each day of use,filtered and degassed prior to the SPR experiments. The target protein(GST-KEAP1) was anchored on a CM5 chip via a GST labelling kit (GEHealthcare, Sweden)³⁴, where a polyclonal goat anti-GST antibody wasimmobilized on a CM5 sensor chip by the amine coupling method 1.

SPR Level-1 (1-point HTS): The SPR Level-1 screening was carried out aspreviously reported³⁵. First, we prepared 10 mM d6-DMSO stock solutionsof the 590 compounds which were procured in powder form. 20 μM samplesof the compounds were made by diluting the stock compounds in runningbuffer with 0.5 mM TCEP and 1% DMSO. The anti-GST immobilizing chip wassaturated with GST at the reference channel and GST-KEAP1 at the targetchannel with resonance unit (RU) values of 750-800 for the GST and2,000-3,000 for GST-KEAP1. Binding of compounds to the immobilizedprotein was monitored for 60 seconds in both the association anddissociation phase. Additional injection of the running buffer wasperformed after every compound binding. All binding signals(RU_(max)=16-29 RU, 1:1 stoichiometry) were corrected for the signalsfrom the reference channel and buffer blank. Compounds were classifiedas an SPR Level-1 hit if the condition RU>4 was satisfied. Thiscriterion was based on the positive control (iKeap1, RU=4.65±0.74).

SPR Level-2 (5-point HTS of the SPR Level-1 hits): The hits from the SPRLevel-1 assay were re-screened at five different compound concentrations(0.5, 1, 5, 10 and 20 μM), in running buffer with 0.5 mM TCEP and 1%DMSO, at a rate of 30 μL/min. The hits were classified as hits if theyproduced a concentration dependent SPR response and an RU value >4 at acompound concentration of 20 μM.

SPR Level-3 (SPR experiments of selected SPR Level-2 hits): 23 out ofthe 69 SPR Level-2 hits were chosen for Level-3 analysis. Given thelow-throughput of the Level-3 SPR assay, we chose a subset of the SPRLevel-2 hits, which included the displacers from the Level-3 FP assay,the compounds that were tested by NMR, and select SPR Level-2 hits. SPRexperiments were carried out in which the target protein (GST-KEAP1) wascaptured and regenerated in each compound cycle. All SPR data processingand analyses were performed using the BiaEvaluation software (version3.0). For steady-state binding, the R_(eq) signal was plotted againstthe analyte concentration and fitted to the one-site or the biphasicbinding model (see Table 4) via the Levenberg-Marquardt algorithm usedby the BiaEvaluation software. The one-site binding model is given bythe equation

R _(eq)=(R _(max) *C)/(K _(d) +C)+b,  (1)

where R_(eq) is the SPR signal at equilibrium, R_(max) is the SPR signalat saturation of the binding mode, K_(d) is the dissociation constant ofthe compound, b is the offset, and C is the concentration of thecompound. The biphasic binding model is given by the equation

R _(eq)=(R _(max,1) *C)/(K _(d,1) +C)+R _(max,2) *C/(K _(d,2)+C)+b,  (2)

where R_(eq) the SPR signal at equilibrium, R_(max,1) and R_(max,2) arethe SPR signals at saturation of the two binding modes, K_(d,1) andK_(d,2) are the dissociation constants of the compound corresponding tothe two binding modes, b is the offset, and C is the concentration ofthe compound.

Standard errors of the estimated K_(d) values were computed with theBiaEvaluation software, which computes them via the diagonal elements ofthe covariance matrix and the residual. The software operates based onthe equations found on page 378 in the book ‘Receptor-LigandInteractions: A Practical Approach’³⁶.

Ligand-Detected NMR Experiments

The differential line broadening (DLB) experiments serve as simpleone-dimensional experiments, where the proton signal of the ligand ismonitored. The ligand concentration exceeds the receptor concentration(e.g. 10-20-fold) in this experiment and broadening of the resonancefrequencies in presence of the receptor is a consequence of ligandmolecules shuttling between free and bound states. DLB manifests as abroadening of the ligand resonance due to binding a protein. The ligandis in equilibrium between the free and protein-bound states dictated bythe equilibrium constant. DLB is the result of the change in relaxationrate and the difference in chemical shift of the bound ligand. In theSTD experiments, a region of the spectral space (−1 to 0.5 ppm) that hasresonances from the receptor but not the ligand is selectivelysaturated. Resonances from methyl bearing amino acids (Ile, Leu, Val)often populate this region of the spectral space. This saturation istransferred to the rest of the protein and eventually to the boundligand by spin diffusion (NOE). In the implementation of STD, twoone-dimensional spectra are recorded in an interleaved fashion. In thefirst experiment neither the receptor nor the ligand is saturated(off-resonance) and in the second the receptor is selectively saturated(on-resonance). Spectra of free ligand are observed in both experiments.However, if the ligand transiently binds to the receptor then thesaturated receptor will transfer magnetization to the ligand. Thistransfer will be reflected as reduced intensity in the on-resonancesaturated spectrum compared to the off-resonance saturation. The resultsare often presented as a difference spectrum between the on andoff-resonance saturation experiments. The appearance of ligandresonances in the difference spectrum is indicative of ligand binding.Measurement of the transverse relaxation rate of the ligand is anothercomplementary strategy to detect ligand binding to a receptor. The freeligand behaves like a small molecule and experiences slow transverserelaxation, however transient binding to the receptor enhances thetransverse relaxation rate of the ligand. Thus, an increased transverserelaxation rate in presence of a receptor directly indicates binding tothe receptor. In the experimental setup a series of 1D experiments wherethe coherences of ligand spends increasing amounts of time in thetransverse plane is recorded. Ligands that engage the protein will relaxfaster than unbound ligand. We refer to these experiments as hear asCPMG-R2 or CPMG experiments. While any of these experiments are inprinciple sufficient to demonstrate ligand binding, false positives foreither of these experiments have been reported. However, a detection ofa false positive hit is highly unlikely if all three experimentsindicate binding, which is the case with all the hits reported here.

All the ligand-detected experiments were performed with 50 μM compoundalone or in presence of 5 μM KEAP1 (without the GST tag) in NMR buffer(phosphate saline buffer supplemented with 5% DMSO-d₆ and 4 mMdeuterated DTT at pH 7.4) unless otherwise noted. For iKeap1 and iKeap2,the protein concentration was kept at 2.5 μM due to tight binding. ¹H 1Dspectra of the compounds were recorded in the absence and in thepresence of KEAP1 to assess line broadening. Saturation transferdifference spectra of the compounds in presence of KEAP1 were recordedwith 3 second saturation time on (0 ppm) and off (−20 ppm) resonance,respectively. The relaxation rate of the compounds was measured in theabsence and the presence of KEAP1 with a series of ¹H 1D experimentswith CPMG-based transverse relaxation time filters of various lengths: 1ms, 25 ms, 50 ms, 100 ms, 300 ms, 500 ms and 800 ms. Data were analysedand visualized in Matlab (MathWorks, MA).

Protein-Detected NMR Experiments

The cleaved mouse KEAP1 Kelch domain (residues 322-624) consists of 308amino acids with close to 300 detectable amide resonances. Therefore,correlating chemical shift perturbations of small molecule inhibitors toperturbations introduced by NRF2 would have been prohibitively difficultin ¹H-¹⁵N HSQC spectra without full backbone assignment. Our aim herewas to rely on methodology that can be quickly and easily implementedeven for very large proteins for which backbone assignment might not befeasible. Indeed, with a molecular weight of 33.7 kDa, the mouse KEAP1Kelch domain (322-624) is already on the larger side for NMR backboneassignment. To overcome the spectral crowding in a ¹H—¹⁵N HSQC spectrumand minimize problems due to low ligand solubility, we implemented a¹H—¹³C TROSY-HMQC experiment coupled with fast data acquisition. For theprotein detected ¹H—¹³C HMQC experiments a sample of KEAP1 which isselectively ¹H and ¹³C labelled at the methyl groups of isoleucine,leucine, and valine residues, in an otherwise deuterated background wasused. This labelling strategy is referred to as ILV labelling.ILV-labelled samples of KEAP1 were prepared by culturing BL21(DE3) cellscontaining a plasmid for GST-KEAP1, in perdeuterated M9 medium with 1 g¹⁵N—NH₄Cl and 2 g ²H-culturing-¹²C-glucose in ²H₂O. One hour beforeinduction with IPTG, 330 mg/L 2-(¹³C)methyl-4-(²H₃)-acetolactate(precursor for leucine and valine) was added. Prior to that theacetolactate was activated as previously described³⁷. 20 min beforeinduction 75 mg/L ¹³C/¹H methyl—and otherwise deuterated—ketobutyratesodium (precursor for isoleucine) was added. The use of acetolactateresulted in stereospecific ¹H—¹³C labelling of only one of the leucine(λ2) and valine (γ2) methyl groups as previously described³⁷. Theprotein was purified as described above. The GST-tag was cleaved bypreScission protease cleavage and the free mouse KEAP1 Kelch domain waseluted in NMR buffer from a size exclusion column. All NMR measurementsfor the ILV-labelled KEAP1 were performed at a protein concentration of5 μM. The protein concentration was kept low to account for poorsolubility (for NMR) of some of the compounds. The concentrations of thecompounds were 50 μM, except for iKeap1 and iKeap2, where theconcentrations were 25 μM, due to poor solubility.

Given the low concentration of the protein, we used the methyl SOFASTmethyl TROSY with 46 ms and 18 ms acquisition times in the direct andindirect dimensions, respectively³⁸. The spectral width was set to 14ppm (¹H) and 20 ppm (¹³C) in the direct and indirect dimensions,respectively and the spectrum was recorded at 298 K on an 800 MHz Brukerspectrometer equipped with an AVANCE III console and a cryogenicallycooled probe. A 4.5 ms Pc9_4_90.1000 pulse was used for selectiveexcitation of the methyl ¹H resonances and a 1.2 ms Rsnob. 1000 pulsewas used to selectively refocus proton chemical shift evolution and¹H—¹³C J-coupling during ¹³C chemical shift evolution. Proper choice andcalibration of the excitation and refocusing pulses is crucial to avoidperturbing the water signal, which can significantly lower theachievable signal to noise. Fast data acquisition was achieved with a150 ms recycling delay, which allowed for the recording of experimentswith 512 scans in 5 hours.

Detecting Aggregation Using Dynamic Light Scattering (DLS)

To test the potential aggregation of hits we used DLS experiments. Theexperiments were performed on a ZS90 Zetasizer instrument (MalvernPanalytical, UK). Measurements were done in triplicate with 10 scans perrun (100 s). The compounds were used at 20 μM concentration in runningbuffer (10 mM HEPES pH 7.5, 50 mM NaCl, 0.1% (v/v) Tween20 with orwithout 0.5 mM TCEP, 2% DMSO) which was filtered before usage. The 20 μMworking solution was made from a 1 mM stock of the compound in DMSO. Thedata was analysed by the built-in software. Compounds were classified asaggregated when the radius of the measured particles was above theminimum colloidal aggregate size (for small molecules) of 50 nm³⁹.

In addition, the solubility of iKeap1, our most potent displacer, wasanalysed with an NMR solubility assay based on a technique describedpreviously⁴⁰. We made individual samples of iKeap1 at variousconcentrations (in PBS buffer, pH 7.4) ranging from 5 μM to 30 μM andmeasured the 1D NMR spectrum of each sample with identical experimentalconditions. The resonances of iKeap1 were then integrated and plotted asa function of concentration. The plot shows a linear trend (R²=0.996)indicating that iKeap1 does not aggregate in this concentration range(FIG. 10 ).

Excluding Interference from PAINS

PAINS comprise 480 markers initially identified as moieties postulatedto cause interference in experimental high-throughput screens⁴¹. PAINScompounds are often found in the databases commonly used for in silicoscreens, and the user should be cognizant of the fact that a potentialhit could harbour a PAINS sub-structure. However, it should also benoted that certain PAINS-like aspects can be mitigated by judicious useof medicinal chemistry, and some aspects of PAINS could have no effect,depending on the target of choice and/or the experimental assaysused^(42,43). Attention should be paid to identifying and rigorouslycharacterizing any PAINS compounds amongst the hits identified in an insilico screen.

Two of the hit compounds (iKeap2 and iKeap7) reported in this manuscriptharbour PAINS substructures. We performed additional experiments toconfirm that iKeap2 and iKeap7 are not false positives due to assayinterference. Primarily, we used 1) DLS to confirm that all thecompounds shown here do not aggregate at the concentrations used in thevarious experiments (Table 6), 2) ligand-detected NMR experiments, STDand CPMG, performed with a 10-fold excess of the compound to show thatiKeap2 and iKeap7 bind KEAP1 in a reversible manner (FIGS. 1A-1H and5A-5F), and 3) protein-observed ¹H—¹³C HMQC experiments to show thatboth iKeap2 and iKeap7 engage KEAP1 in a specific manner at the NRF2binding site and do not aggregate the protein (FIGS. 7A-7H and 9A-9H).In the event these compounds caused the KEAP1 to aggregate, all theresonances will be broadened, which was not the case here.

Statistics and Reproducibility

Screening size 100K: minimum: −10.3; maximum: −11.6; median: −10.4; Q₁:−10.4, Q₃: −10.6. Screening size 1M: minimum: −10.9; maximum: −12;median: −11; Q₁: −11.1, Q₃: −11.3. Screening size 10M: minimum: −11.675;maximum: −12.3; median: −11.5; Q₁: −11.4, Q₃: −11.5. Screening size100M: minimum: −11.8; maximum: −12.6; median: −11.9; Q₁: −11.8, Q₃:−12.1. Screening size 1B: minimum: −12.3; maximum: −13.4; median: −12.4;Q₁: −12.3, Q₃: −12.6.

Data Availability

The ready-to-dock library from Enamine is freely available online on thehomepage of VirtualFlow at http://virtual-flow.org/real-library. Sourcedata of FIGS. 3A-3H, 4A-4H and 5A-5F is available online athttps://doi.org.

Code Availability

VirtualFlow is mainly written in Bash (a Turing complete commandlanguage), which not only makes it simple for anyone to modify andextend the code, but also has essentially no computational overhead andis readily available in any major Linux distribution. The code forVirtualFlow is freely available on https://github.com/VirtualFlow,distributed under the GNU GPL open-source license. The primary homepagefor end users where additional resources including documentation, ligandlibraries, tutorials and video demonstrations is available athttps://www.virtual-flow.org. The external docking programs discussedhere are available as follows: AutoDock Vina is available athttp://vina.scripps.edu, QuickVina 2 and QuickVina-W athttps://qvina.github.io, Vina-Carb athttp://glycam.org/docs/othertoolsservice/download-docs/publication-materials/vina-carb,Smina at https://sourceforge.net/projects/smina, AutoDockFR athttp://adfr.scripps.edu and VinaXB athttps://github.com/ssirimulla/vinaXB.

Supplementary Information

A. KEAP1 Binders from the Literature

Table 2 lists the experimentally verified binders to the NRF2-bindingdomain of KEAP1 found in [19, 15, 29, 11], which were added to theprimary virtual screening for the purpose of further validation ofVirtualFlow. The threshold docking score (resembling the free energy ofbinding ΔG) for the top 10% of the compounds is −8.6 kcal/mol. All 17 ofthe compounds have a predicted docking score above that threshold,indicating that the docking procedure in the primary virtual screeninghas worked well, despite the use of the lowest possible dockingaccuracy.

TABLE 2 Docking IC₅₀ K_(d) Score Compound SMILES [μM] [μM] [kcal/mol] C1O═C(O)[C@@H]1CCCC[C@@H]1C(═O) 3 −8.7 N3CCe2ccccc2C3Cn5c(═O)c4ccccc4c5═OC2 O═C(O)[C@H]1CCCC[C@H]1C(═O)] 2.3 −9.0 N1CCc2ccccc2[C@@H]1CN1C(═O)c2ccccc3c(═O) C3 O═C(O)[C@H]1CCC[C@H]1C(═O) 2.2 −9.8N3CCc2ccccc2[C@H]3Cn5e(═O) c4ccccc4c5═O C4 O═C(O)[C@H]1CC[C@H]3C(═O) 8.0−9.3 N3CCc2cccccc2[C@H]3Cn5c(═O) c4ccccc4c5═O C5O═C(O)[C@H]1C[C@H]1C(═O) 20.8 −9.6 N3CCe2cccc2[C@H]3Cn5c(═O) c4cccc4c5═OC6 O═C(O)[C@H]1CCNC[C@H]1C(═O) 69.7 −8.7 N3CCe2ccccc2[C@H]3Cn5c(═O)c4ccccc4c5═O C7 O═C(O)[C@H]1CCCC[C@H]1C(═O) 1.1 −9.5N1CCc2ccccc2[C@H]1CN1Ce2ccccc2C1═O C8 O═C(O)[C@H]1CCCC[C@H]1C(═O) 1.2−9.1 N3CCc2ccccc2[C@H]3CN4C(═O)CCC4═O C9 O═C1c2ccccc2C(═O) 7.4 −10.8N1C[C@@H]1c2ccccc2CCN1C (═O)[C@@H]1CCCC[C@@H]1c1nno[

C10 Cc1cc(C)c(C)c(S(═O)(═O)Nc2ccc 0.14 −10.5(N3CC[C@@H]1C(═O)O)C3)c3cccc23c1C C11CC(C)c1ccc(S(═O)(═O)Nc2cc(Sc3ncn(nH)3)c(O) 3.9 −9.4 c3cccc23jcc1 C12O═C(O)c4cccc3ccc(C═c2c(═O)(cH)n(c1cccc(C) 15.2 −8.8 cc1)c2═O)c3)c4C1 C13Cc4cccc1NC(═O)Cn4c(═O)sc 10.4 −16.0 (═cc2cccn2C3CCCC(C(═O)O)C3)C4═O C14CN(Cc1cc[C@H](CC(═O)O)c2ccc3c(c2) 3.4 −8.7 non3Cccc)ClS(C)(═O)═O C15CN(Cc1cc[C@H](OC(═O)O)c2cc3c(c2)mm3C) 0.27 −9.7 ccc(C)S(═O)(═O)c1cccc1C16 Cc3ccc([C@H](CC(═O)O)c1ccc2c(c1nnn3C) 0.015 −10.8nn3CN3C[C@@H](CcOc4ccccc4S5(═O)═O C17 COc1ccccS(═O)(═O)Nc3ccc(NS(═O)(═O)2.7 −9.9 c3ccc(OC)cc3)c3ccccc23)cc1

indicates data missing or illegible when filedPreviously identified binders to the NRF2-binding domain of KEAP1. Shownare the SMILES-formatted chemical structures, the reported IC₅₀ values,and the predicted docking scores from the primary virtual screen. Thegoal of the primary virtual screen was to distinguish binders fromnon-binders for demonstration purposes, and the stringency was set tothe lowest possible level.

In FIG. 3A the crystal structure which was used for the virtualscreening procedure is shown, as well as the structure of iKeap1 (FIG.3C). The structure of iKeap1 is similar to a previously publishedinhibitor of KEAP1, shown as compound C17(N,N′-Naphthalene-1,4-Diylbis(4-Methoxybenzenesulfonamide) in Table 2and FIG. 3D [19]. The predicted docking position for iKeap1 (FIG. 1A) isvery similar to the co-crystal structure of compound C17 (FIG. 3D 5 d).These similarities further substantiate that iKeap1 binds to theKEAP-1-NRF2 interface.

B. Experimental Validation Experiments

The SPR Level-2 compounds can be filtered using several characteristics,such as their suitability for medicinal chemistry, predicted pan assayinference (PAINS), or ability to displace the NRF2 peptide. In Table 3we are listing the hit compounds which are presented in the manuscript.Also included in the table are all the other hits which are able todisplace the NRF2 peptide, which do not harbour any PAINS substructures,and which do not contain other problematic substructures (such asazo-dye compounds or compounds which are unsuitable for medicinalchemistry). It should be noted that, in addition to the compounds listedhere, our experimental validation identified other potent displacers andbinders which were filtered out due PAINS and/or non-drug likeproperties.

TABLE 3 ID SMILES FP Assay BLI Assay iKeap1Cc3ccc(S(═O)(═O)Cn2nc3nc4ccccc4cc3ns2OS active active(═O)(═O)c2ccc(O)cc2cc1 iKeap2 O═C(O)c4ccc(NC2═C/C(═N/S(═O)(═O) activeactive c3ccc4ccccc4e3nc3ccccc3C2═Occ) iKeap7OC3═NN(c2ccccc2)C(═O)/C1═/s4ccc active active (-c2cc(C(═O)(═O)ccc2C 

iKeap8 O═C(Nc1cccc active active (-c2ccn[nH]3 

[C@@H]3C[C@H]2CCCC[C@H]2N1C (═O)c1ccc2ccccc2c1 iKeap9O═S(═O)c(N═C1CCCCCN1)C1═CC═C25C not active not active (NS(═O)(═O)C3═CC═C4C═CC═CC4═C3)═NC2═C1 iKeap12 O═C(c3ccccc 

 C1═C[C@@H]2[C@@H]3C(═O)N active active(c4ccccSccccc54)C(═O)[C@@H]3[C@@H](C4═O) c3cccCl)cc3)N2C═O1 iKeap22O═c1cc(-c2cge(O)cc2nc2c1c(O)cc1c2[C@H] not active active(c2ccc3ncccc3c2)CC(═O)O1 iKeap27Cc1ccc(C)c(N2C(═O)c3ccc(-c4nc(-c5ccc[nH]nnc6c5) not active activenn4)cc3C2═Ojc1 iKeap33 O═S(═O)Nc 

 2cccc2nc1N1CCC[C@@H] not active active (c2nc3ccccc3[nH]3C1)c1ccccc1iKeap41 O═C(c1cc(nc2ccc3 

 2)OCCO3)MC2CCCCC21) not active active N1CCC(CC2CCCCC2)CC1 iKeap48CC(═O)S/C(nC)c1ccc(Cc2nc3c 

 C(═O)c2ccccc3C not active active (═O)cc4c32sc2c[nH]C(═O)c3ccccc1C3═OiKeap73 C[C@H]1CC2cnc3nccs(N4CCO[C@@H](CN5C(═O) not active activec5ccccc6C5═O)C4)c23)C1 iKeap74 C[C@@H}1Oc2ccc(C(═O)C3CCN(C(═O) notactive active [C@H]4C[C@H]4c4ccc5cccc45)CC3)cc2NC3═O iKeap75Cc3cccn2c(═O)c3c(nc32)N1CCCCC[C@H]3[C@H] not active active(C3)C(═O)N(Cc2ccccc2C)C(═O)[N═C]O

indicates data missing or illegible when filedSPR Level-2 hit compounds, which were also tested experimentally via afluorescence polarization (FP) assay and BLI experiments for theirability to displace the NRF2-peptide. All compounds shown in this tablehave also been verified by protein detected NMR experiments (STD, CPMG,DLB, ¹H—¹³C HMQC), or have passed additional filters to remove compoundswith problematic substructures (regarding pan assay interference orsuitability for medicinal chemistry).

Two of the hit compounds (iKeap2 and iKeap7) contain PAINS alerts. Wehave carried a suite of orthogonal experiments to show that thecompounds are indeed true and specific binders, and are not artifactsdue to pan assay interference. DLS and 1D NMR show the compounds are notaggregating, and they are not similar to any known aggregators asassessed by the Tanimoto similarity measure [IDT⁺ 15]. STD-NMR and R2measurements show the compounds are not covalently binding. The FP assayshows that the compounds bind at the targeted peptide-binding site bydisplacing it. The FP assay is done in the presence of BSA and toaccount for non-specific binding. Protein-observed NMR experiments(¹H—¹³C-HMQC) clearly shown the compounds site-specifically engage KEAP1in a manner similar to NRF2. The protein-detected NMR experiments showthat both iKeap2 and iKeap7 do not aggregate the protein (KEAP1).

TABLE 4 K_(d, 1) K_(d, 2) K_(d, 3) K_(d) ^(average) SD Compound (nM)(nM) (nM) χ₁ ² χ₂ ² χ₃ ² (nM) (nM) ikeap1* 114 178 85 0.026 0.404 0.033125.667 47.585 iKeap2 158 263 334 0.031 0.333 0.265 251.667 88.546iKeap7 23 15 16 0.0305 0.306 0.019 18.000 4.359 iKeap8* 176 115 1870.118 0.130 0.397 159.333 38.786 iKeap9* 180 187 190 0.278 0.242 0.158185.667 5.132 iKeap22* 62 51 52 0.082 0.109 0.051 55.000 6.083Kd values which were determined by SPR Level-3 experiments of thecompounds shown in more detail above (FIGS. 1A-1H, 4A-4H and 5A-5F). Foreach compound three independent experiments were carried out, givingrise to three K_(d,i) and χi² values, where i indicates the independentexperiment. Compounds marked with an asterisk were determined by fittingthe biphasic binding model given by equation (6) to the experimentaldata,

TABLE 5 Compound IC₅₀ (μM) Standard Error [μM] iKeap1 0.258 0.037 iKeap22.7 0.6 iKeap7 38.2 3.9 iKeap8 14.2 4.0IC₅₀ values and the associated errors which were determined by theLevel-2 FP experiments are shown here for the compounds highlighted inthe manuscript. Representative curves for these FP hits are shown inmore detail in the manuscript (FIGS. 1A-1H, 4A-4H and 5A-5F)

TABLE 6 Radius Standard Compound [nm] Error [nm] buffer 8.781 6.782iKeap1 2.306 2.534 iKeap2 12.492 2.008 iKeap7 3.663 3.449 iKeap8 3.3401.117 iKeap9 1.862 0.603 iKeap12 44.780 31.646 iKeap22 2.547 1.890iKeap27 2.643 3.017 iKcap33 1.585 1.779 iKeap41 4.446 0.887 iKeap4825.579 31.497 iKeap73 2.510 1.583 iKeap74 3.034 1.972 iKeap75 3.6121.527DLS data for the hit compounds listed in Table 3 showing that thesecompounds are not aggregating. According to [IDT+15] colloidalaggregates of compounds have radii in the range of 50-800 nm. Allmeasurements were done in triplicates and the average and standard erroris shown here.

REFERENCES

-   1. DiMasi, J. A., Grabowski, H. G. & Hansen, R. W Innovation in the    pharmaceutical industry: New estimates of R&D costs. J. Health Econ.    47, 20-33 (2016).-   2. Lyu, J. et al. Ultra-large library docking for discovering new    chemotypes. Nature 566, 224-229 (2019).-   3. Zhang, S., Kumar, K., Jiang, X., Wallqvist, A. & Reifman, J.    DOVIS: an implementation for high-throughput virtual screening using    AutoDock. BMC Bioinformatics 9, 126 (2008).-   4. Jiang, X., Kumar, K., Hu, X., Wallqvist, A. & Reifman, J. DOVIS    2.0: an efficient and easy to use parallel virtual screening tool    based on AutoDock 4.0. Chem. Cent. J 2, 18 (2008).-   5. Hassan, N. M., Alhossary, A. A., Mu, Y & Kwoh, C.-K.    Protein-Ligand Blind Docking Using QuickVina-W With Inter-Process    Spatio-Temporal Integration. Sci. Rep. 7, 15451 (2017).-   6. Bohacek, R. S., McMartin, C. & Guida, W. C. The art and practice    of structure-based drug design: A molecular modeling perspective.    Med. Res. Rev. 16, 3-50 (1996).-   7. Yonchuk, J. G. et al. Characterization of the Potent, Selective    Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2    H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary    Oxidative Stress. J Pharmacol. Exp. Ther 363, 114-125 (2017).-   8. Pallesen, J. S., Tran, K. T. & Bach, A. Non-covalent    Small-Molecule Kelch-like ECH-Associated Protein 1-Nuclear Factor    Erythroid 2-Related Factor 2 (Keap1-Nrf2) Inhibitors and Their    Potential for Targeting Central Nervous System Diseases. J. Med.    Chem. 61, 8088-8103 (2018).-   9. Davies, T. G. et al. Monoacidic Inhibitors of the Kelch-like    ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor    2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency    Identified by Fragment-Based Discovery. J Med. Chem. 59, 3991-4006    (2016).-   10. Cuadrado, A. et al. Therapeutic targeting of the NRF2 and KEAP1    partnership in chronic diseases. Nat. Rev. Drug Discov. 18, 295-317    (2019).-   11. SLURM. https://slurm.schedmd.com. Accessed: 2018-Dec.-22.-   12. Moab/TORQUE. http://www.adaptivecomputing.com. Accessed:    2018-Dec.-22.-   13. PBS Professional. http://www.pbspro.org. Accessed: 2018-Dec.-22.-   14. LFS.    https://www.ibm.com/us-en/marketplace/hpc-workload-management.    Accessed: 2018-Dec.-22.-   15. SGE. http://gridscheduler.sourceforge.net. Accessed:    2018-Dec.-22.-   16. Sterling, T. & Irwin, J. J. ZINC 15—Ligand Discovery for    Everyone. J Chem. Inf Model. 55, 2324-2337 (2015).-   17. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and    accuracy of docking with a new scoring function, efficient    optimization, and multithreading. J. Comput. Chem. 31, 455-61(2010).-   18. Alhossary, A., Handoko, S. D., Mu, Y & Kwoh, C.-K. Fast,    accurate, and reliable molecular docking with QuickVina 2.    Bioinformatics 31, 2214-2216 (2015).-   19. Koes, D. R., Baumgartner, M. P. & Camacho, C. J. Lessons Learned    in Empirical Scoring with smina from the CSAR 2011 Benchmarking    Exercise. J Chem. Inf Model. 53, 1893-1904 (2013).-   20. Ravindranath, P. A., Forli, S., Goodsell, D. S., Olson, A. J. &    Sanner, M. F. AutoDockFR: Advances in Protein-Ligand Docking with    Explicitly Specified Binding Site Flexibility. PLOS Comput. Biol.    11, e1004586 (2015).-   21. Koebel, M. R., Schmadeke, G., Posner, R. G. & Sirimulla, S.    AutoDock VinaXB: implementation of XBSF, new empirical halogen bond    scoring function, into AutoDock Vina. J. Cheminformatics 8, 27    (2016).-   22. Nivedha, A. K., Thieker, D. F., Makeneni, S., Hu, H. &    Woods, R. J. Vina-Carb: Improving Glycosidic Angles during    Carbohydrate Docking. J Chem. Theory Comput. 12, 892-901 (2016).-   23. Amaro, R. E. et al. Ensemble Docking in Drug Discovery.    Biophys. J. 114, 2271-2278 (2018).-   24. Houston, D. R. & Walkinshaw, M. D. Consensus Docking: Improving    the Reliability of Docking in a Virtual Screening Context. J. Chem.    Inf Model. 53, 384-390 (2013).-   25. Marcotte, D. et al. Small molecules inhibit the interaction of    Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.    Bioorg. Med. Chem. 21, 4011-4019 (2013).-   26. Andrei, S. A. et al. Stabilization of protein-protein    interactions in drug discovery. Expert Opin. Drug Discov. 12,    925-940 (2017).-   27. Ragoza, M., Hochuli, J., Idrobo, E., Sunseri, J. & Koes, D. R.    Protein-Ligand Scoring with Convolutional Neural Networks. J Chem.    Inf Model. 57, 942-957 (2017).-   28. Reymond, J. The Chemical Space Project. Acc. Chem. Res. 48,    722-730 (2015).-   29. ChemAxon. JChem Suite.    https://chemaxon.com/products/jchem-engines. Accessed: 2020-Jan.-10.-   30. O'Boyle, N. M. et al. Open Babel: An open chemical toolbox. J    Cheminformatics 3, 1-14 (2011).-   31. Enamine. REAL Library. https://enamine.net/library-synthesis    real-compounds. Accessed: 2020-Jan.-10.-   32. Morris, G. M. et al. AutoDock4 and AutoDockTools4: Automated    docking with selective receptor flexibility. J Comput. Chem. 30,    2785-2791 (2009).-   33. SciPy developers. SciPy. https://www.scipy.org. Accessed:    2020-Jan.-10.-   34. Surface Plasmon Resonance. vol. 627 (Humana Press, 2010).-   35. Hämäläinen, M. D. et al. Label-free primary screening and    affinity ranking of fragment libraries using parallel analysis of    protein panels. J Biomol. Screen. 13, 202-209 (2008).-   36. Receptor-Ligand Interactions A Practical Approach. (Oxford    University Press, 1992).-   37. Gans, P. et al. Stereospecific Isotopic Labeling of Methyl    Groups for NMR Spectroscopic Studies of High-Molecular-Weight    Proteins. Angew. Chem. Int. Ed. 49, 1958-1962 (2010).-   38. Lu, M. et al. Discovery of a Keap1-dependent peptide PROTAC to    knockdown Tau by ubiquitination-proteasome degradation pathway. Eur    J Med. Chem. 146, 251-259 (2018).-   39. Irwin, J. J. et al. An Aggregation Advisor for Ligand    Discovery. J. Med. Chem. 58, 7076-7087 (2015).-   40. Laplante, S. R. et al. Compound Aggregation in Drug Discovery:    Implementing a Practical NMR Assay for Medicinal Chemists. J Med.    Chem. 56, 5142-5150 (2013).-   41. Baell, J. B. & Holloway, G. A. New substructure filters for    removal of pan assay interference compounds (PAINS) from screening    libraries and for their exclusion in bioassays. J. Med. Chem. 53,    2719-2740 (2010).-   42. Baell, J. B. & Nissink, J. W. M. Seven Year Itch: Pan-Assay    Interference Compounds (PAINS) in 2017—Utility and Limitations. ACS    Chem. Biol. 13, 36-44 (2018).-   43. Capuzzi, S. J., Muratov, E. N. & Tropsha, A. Phantom PAINS:    Problems with the Utility of Alerts for Pan-Assay INterference    CompoundS. J Chem. Inf Model. 57, 417-427 (2017).

Example 2: NQO1 Assay

NQO1 (NAD(P)H Quinone Dehydrogenase is a target gene of NRF2 and hasbeen used to monitor the activity of the NRF2 pathway. See, for example,A. T. Dinkova-Kostova and P. Talalay, NAD(P)H:quinone acceptoroxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme andexceptionally versatile cytoprotector, Arch. Biochem. Biophys. (2010),vol. 501, pp. 116-123. NQO1 is a ubiquitous flavoenzyme that catalyzesthe two-electron reduction of quinones to hydroquinones. NQO1 geneexpression and enzymatic activity is known to increase proportionatelywith Nrf2 activation, and this enzyme is routinely used by the field asa NRF2 biomarker for in vitro cell-based assays of compound activity.

In this study, the inventors used a calorimetric assay to quantitate theNQO1 levels. See, for example, H. J. Prochaska and A. B. Santamaria,Direct measurement of NAD(P)H:quinone reductase from cells cultured inmicrotiter wells: a screening assay for anticarcinogenic enzymeinducers, Anal. Biochem. (1988), vol. 169, pp. 328-336, and J. W. Faheyet al., A. T. Dinkova-Kostova, K. K. Stephenson, P. Talalay, The“Prochaska” microtiter plate bioassay for inducers of NQO1, MethodsEnzymol. (2004), vol. 382, pp. 243-258. Specifically, Hepa1c1c7 cellswere exposed in 8 replicates to a vehicle (0.200 DMSO) or 8 serialdilutions of each compound for 48 h. Cells were then lysed and thespecific activity of NQO1 was determined using menadione as a substrate.Results are shown in FIG. 11 and summarized in Table 7.

TABLE 7 Publication IDs: iKeap1 iKeap4 iKeap18 iKeap8 iKeap7 iKeap2iKeap17 HMS IDs: uM KN39 KN67 KN53 KE157 KX29 KS7 KY190 0 1 1 1 1 1 1 10.156 1.19 1.06 1.09 1.07 1.06 1.13 1.33 0.313 1.28 1.1 1.09 1.04 1.081.2 1.44 0.625 1.41 1.21 1.19 1.06 1.13 1.33 1.52 1.25 1.68 1.18 1.181.11 1.17 1.49 1.61 2.5 2.1 1.27 1.29 1.08 1.3 1.91 1.6 5 2.66 1.47 1.391.11 1.44 2.75 1.7 10 3.53 1.75 1.58 1.16 1.65 6.08 1.51 20 4.18 2.21.79 1.21 2.08 1.43 CD (uM) 2 15 40 (extp) 20 2.7 Note: Compound KS7 wastoxic at concentrations higher than 2.5 uM

All patents and other publications identified herein are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentdisclosure. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior disclosure orfor any other reason. All statements as to the date or representation asto the contents of these documents are based on the informationavailable to the applicants and do not constitute any admission as tothe correctness of the dates or contents of these documents.

What is claimed is:
 1. A method of inhibiting Kelch-like ECH-associatedprotein 1 (KEAP1), the method comprising contacting KEAP1 with acompound selected from the following: (i) compounds of Formula (I):

wherein: R¹ is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;R² is substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocyclyl; R³ ishydrogen, —OR^(A), —SR^(A), or —N(R^(A))₂; and each R^(A) independentlyis H, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, or substituted orunsubstituted cyclyl, or a pharmaceutically acceptable salt, solvate,hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopicallylabeled derivative, or prodrug thereof; (ii) compounds of Formula (II):

wherein: A is substituted or unsubstituted arylene, substituted orunsubstituted biarylene, or substituted or unsubstituted heteroarylene;R⁴ is substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocyclyl; and R⁵ issubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocyclyl; or apharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof; (iii) compounds of Formula (III):

wherein: each A is independently substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁶ is substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted heterocyclyl; and R⁷ is substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted heterocyclyl; or a pharmaceuticallyacceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer,stereoisomer, isotopically labeled derivative, or prodrug thereof; or(iv) a compound selected from Group A, wherein the Group A comprises thecompounds:

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.
 2. (canceled)
 3. (canceled)
 4. The method of claim 1,wherein the KEAP1 is in a cell and the method comprised administeringthe compound to the cell.
 5. The method of claim 4, wherein saidadministering to the cell is in vitro.
 6. The method of claim 4, whereinsaid administering to the cell is in vivo.
 7. The method of claim 6,wherein said administering to the cell is in a subject having ordiagnosed with a disease associated with dysfunction of Nrf2-KEAP1 axisor a disease associated with Nrf2-KEAP1 interaction.
 8. (canceled)
 9. Amethod treating a disease associated with dysfunction of the Nrf2-KEAP1axis or a disease associated with Nrf2-KEAP1 interaction in a subject inneed thereof, the method comprising administering to a subject in needthereof a therapeutically effective amount a compound selected from ofthe group consisting of compounds of Formula (I), compounds of Formula(II), compounds of Formula (III), compounds of Group A, or apharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.
 10. (canceled)
 11. The method of claim 9, wherein thedisease is associated with oxidative stress.
 12. The method of claim 11,wherein the disease is selected from the group consisting abdominalaortic aneurysm, acute kidney injury, adult brain glioblastoma, advancedsolid tumors lymphoid malignancies, aging, alcohol sensitivity,allergic, Alport syndrome, Alzheimer's disease, asthma, atopicasthmatics, autism spectrum disorder, autosomal dominant polycystickidney, Barrett esophagus, low-grade dysplasia, brain ischemia, breastcancer or breast neoplasm, cardiovascular risk, cataract surgery,cholelithiasis, cholestasis, chronic hepatitis c, chronic kidneydisease, chronic lymphocytic leukemia, chronic renal insufficiency,chronic schizophrenia, chronic subclinical inflammation, CKD associatedwith type 1 diabetes, cognition, colon cancer, COPD, corneal endothelialcell loss, crohn's disease, cutaneous t cell lymphoma, diabetesmellitus, diabetic nephropathy, diarrhea, endometriosis, environmentalcarcinogenesis, focal segmental glomerulosclerosis, Friedreich's ataxia,healthy, Helicobacter pylori infection, hepatic impairment, healthy,huntington disease, IgA nephropathy, inflammation and pain followingocular surgery, insulin resistance, liver disease, lung cancer, majordepression, melanoma, metabolic syndrome x, mild cognitive impairment,mitochondrial myopathy, multiple sclerosis, neoplasms, nonalcoholicfatty liver or nonalcoholic steatohepatitis, noninsulin-dependent,nonischemic cardiomyopathy, obstructive sleep apnea, ocularinflammation, ocular pain, polymorphism, prediabetes, primary biliarycirrhosis, primary focal segmental glomerulosclerosis (FSGS), prostatecancer, psoriasis, psychosis, pulmonary arterial hypertension (pah),pulmonary hypertension, redox status, rheumatoid arthritis, rhinitis,schistosomiasis, schizophrenia, small lymphocytic lymphoma, subarachnoidhaemorrhage, and type 2 (type 2 diabetes).
 13. The method of claim 9,wherein the subject is a mammal.
 14. The method of claim 9, wherein thesubject is human.
 15. The method of any claim 9, wherein the compound isof Formula (I).
 16. The method of claim 9, wherein the compound is

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof
 17. The method of claim 9, wherein the compound is ofFormula (II).
 18. The method of claim 9, wherein the compound is

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof.
 19. The method of claim 9, wherein the compound is ofFormula (III).
 20. The method of claim 9, wherein the compound is

or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,co-crystal, tautomer, stereoisomer, isotopically labeled derivative, orprodrug thereof
 21. The method of claim 9, wherein the compound is acompound selected from Group A.
 22. The method of claim 9, wherein thecompound has a structure defined by a Simplified Molecular Input LineEntry System (SMILES) selected from the group consisting of:CC1=CC═C(C(═O)NC(═CC2=CC═C(C3=CC═CC([N+](═O)[O—])═C3)O2)C(═O)NCC2CCCO2)C═C1(iKeap 28),O═C(O)c1ccc(NC2=C/C(═N\S(═O)(═O)c3ccc4ccccc4c3)c3ccccc3C2=O)cc1(iKeap2),O═C(O)C(SC1=NC(═O)C2(NN1)c1ccccc1-c1ccccc12)SC1=NC(═O)C2(NN1)c1ccccc1-c1ccccc12(iKeap 24),O═S(═O)(N═C1CCCCCN1)C1=CC═C2SC(NS(═O)(═O)C3=CC═C4C═CC═CC4=C3)=NC2=C1(iKeap9),Cc1cc2oc(=O)cc(COC(═O)[C@H]3CCCN(C(═O)c4ccc5[nH]ncc5c4)C3)c2cc1C(C)C(iKeap20), O═C1OC(c2ccc([N+](═O)[O-])cc2)=N/C1=C\c1cccc2ccccc12(iKeap4), COc1ccc(NS(═O)(═O)c2ccc(/N=C\c3c4ccccc4nc4ccccc43)cc2)nn1(iKeap29),Cc1ccc(C)c(N2C(═O)c3ccc(-c4nc(-c5ccc6[nH]nnc6c5)no4)cc3C2=O)c1(iKeap27),Cc1cccn2c(=O)c3c(nc12)N1CCCCC[C@H]1[C@]1(C3)C(═O)N(Cc2ccccc2C1)C(═O)N═C1O (iKeap75),O═C(Cn1nc(-c2ccccc2)ccc1=O)Nc1cccc(Oc2ccc([N+](═O)[O-])cc2)c1 (iKeap51),O═C(c1ccccc1)C1=C[C@@H]2[C@@H]3C(═O)N(c4cccc5ccccc54)C(═O)[C@@H]3[C@@H](C(═O)c3ccc(C1)cc3)N2C═C1(iKeap12),O═S(═O)(O)c1ccc(/N=N\c2ccc(/N=N\c3ccc(O)c4cccc(S(═O)(═O)O)c34)cc2)cc1(iKeap18),O═C1C[C@H](C(═O)N2CCC[C@H](NC(═O)c3cc4ccccc4o3)C2)c2ccc(F)cc2N1(iKeap26),O═C(NN═CC1=C(O)C([N+](═O)[O—])═CC(C1)=C1)C1=CC(C2=CC═CC═N2)=NC2=CC═CC═C12(iKeap36),Cc1ccc2cc([C@H]3CC(═O)Oc4cc(O)c5c(=O)c(O)c(-c6ccc(O)c(O)c6)oc5c43)c(=O)[nH]c2c1(iKeap52), CC1=NN(c2ccccc2)C(═O)/C1=Cc1ccc(-c2cc(C(═O)O)ccc2C1)o1(iKeap7),O═C1C[C@H](c2ccc(OCCc3ccc4c(c3)CCO4)cc2)c2c(cc(O)c3c(=O)c(O)c(-c4ccc(O)c(O)c4)oc32)O1(iKeap31),O═C(c1ccccc1)c1cc(-c2cc(=O)cc(-c3cc(C(═O)c4ccccc4)c(O)cc3O)o2)c(O)cc1O(iKeap16),CC(C)c1ccc(COc2cccc([C@H]3CC(═O)Oc4cc(O)c5c(=O)c(O)c(-c6ccc(O)c(O)c6)oc5c43)c2)cc1(Ikeap34), O═C1OC(c2cccc3ccccc32)=N/C1=Cc1ccc(-c2cccc(C(F)(F)F)c2)o1(Ikeap13),COc1ccc2occ([C@@H]3CC(═O)Oc4cc(O)c5c(=O)cc(-c6ccc(O)c(O)c6)oc5c43)c(=O)c2c1(IKeap 62),CC1=CC═C(S(═O)(═O)NC2=CC(═NS(═O)(═O)C3=C(C)C═C(C)C═C3C)C3=CC═CC═C3C2=O)C═C1 (iKeap69),Cc1cccn2c(=O)c3c(nc12)N1CCCCC[C@H]1[C@]1(C3)C(═O)N(Cc2ccccc2)C(═O)N═C1O(iKeap 39),CC1=NOC(NS(═O)(═O)C2=CC═C(NC(═O)CC3=COC4=CC═C(C(C)C)C═C34)C═C2)=C1C(Ikeap68),Cc1ccc(N2C(═O)[C@@H]3N═NN(CC(═O)N4N═C5/C(=C/c6ccccc6)CCC[C@@H]5[C@H]4c4ccccc4)[C@@H]3C2=O)cc1(iKeap40),Nc1ccc2cc(S(═O)(═O)O)c(/N═N/c3ccc(/N═N\c4ccc(N)c5cc(S(═O)(═O)O)ccc45)c4ccccc34)c(O)c2c1 (iKeap5),Cc1ccc(/N═N\c2cc(C)c(N)c(/N═N\c3ccc(/N═N\c4cc(C(═O)O)c(O)c(S(═O)(═O)O)c4)c(C)c3)c2N)cc1(iKeap30),COc1ccc(C(═O)C2=C(O)C(═O)N(c3nnc(SCc4cccc5ccccc54)s3)[C@H]2c2cccc(Oc3ccccc3)c2)cc1OC (iKeap56),CC1=NN(C2=CC═CC═C2)C(NC2=CC═C3C(═N2)OC2=NC(NC4=CC(C)═NN4C4=CC═CC═C4)=CC═C2C3C2=CC═C(C1)C([N+](═O)[O-])═C2)=C1 (iKeap35),Cc1ccc(/C═C2\CCC[C@@H]3C2=NN(C(═O)CN2N═N[C@@H]4C(═O)N(c5cccc(F)c5)C(═O)[C@H]42)[C@@H]3c2ccc(C)cc2)cc1(iKeap57),Cc1ccc(Nc2ccc(/N═N\c3ccc(/N=N\c4cccc(S(═O)(═O)O)c4)c4ccccc34)c3cccc(S(═O)(═O)O)c23)cc1 (iKeap6),COc1cc(/N═N\c2ccc(S(═O)(═O)O)cc2)ccc1/N=N\c1ccc2c(cccc2S(═O)(═O)O)c1O(iKeap 11),O═C1C[C@@H](c2ccccc2)CC2=C1[C@@H](c1cccc(Oc3ccccc3)c1)Nc1ccccc1N2(iKeap77),O═S(═O)(O)c1cc(/N=N/c2c(O)ccc3ccccc32)ccc1/C═C\c1ccc(/N═N\c2c(O)ccc3ccccc32)cc1S(═O)(═O)O (iKeap38),O═C(O)c1cc(/N═N\c2ccc(/C═C\c3ccc(/N═N\c4ccc(O)c(C(═O)O)c4)cc3S(═O)(═O)O)c(S(═O)(═O)O)c2)ccc1O(iKeap23),O═C(N/N═C/c1cc([N+](═O)[O-])cc([N+](═O)[O-])c1O)c1cc(-c2ccccc2)nc2ccccc21(iKeap54),O═[N+]([O-])c1ccc(Nc2ccc(Oc3ccc(Nc4ccc([N+](═O)[O—])c5nonc54)cc3)cc2)c2nonc21(iKeap14),Cc1ccc(S(═O)(═O)Oc2nc3nc4ccccc4nc3nc2OS(═O)(═O)c2ccc(C)cc2)cc1 (ikeap1),Cc1nnnn1-c1cccc(NC(═O)c2c3c(nc4ccccc24)/C(═C\c2ccco2)CC3)c1 (iKeap46),O═C1N[C@@H](Cc2nc(-c3cccc(Cn4cnc5ccccc54)c3)no2)C(═O)Nc2ccccc21(iKeap60), Cc1ccnc(NS(═O)(═O)c2ccc(/N═C\c3c4ccccc4nc4cc(C1)ccc34)cc2)n1(iKeap50), O═C(Cc1ccc(C1)cc1)Nc1cccc(-c2ccc3nnc(-c4cccnc4)n3n2)c1(iKeap32),O═C([C@H]1C[C@H]2CCCC[C@@H]2N1c1ncccn1)N1CC═C(c2c[nH]c3cc(F)ccc23)CC1(iKeap67),Nc1ccc2c(O)c(/N═N\c3ccc(/N═N\c4ccc(S(═O)(═O)O)cc4)cc3)c(S(═O)(═O)O)cc2c1/N═N\c1ccc([N+](═O)[O-])cc1 (iKeap55),Cc1cc(/N═N\c2ccc(S(═O)(═O)O)cc2C)ccc1/N═N\c1cc(S(═O)(═O)O)c2ccccc2c1O(iKeap3),O═S(═O)(O)c1cc(O)c2c(c1)cc(S(═O)(═O)O)cc2/N═N\c1ccc(Nc2ccccc2)c2c1cccc2S(═O)(═O)O(iKeap25),O═S(═O)(O)c1cc(/N═N\c2ccc(O)c3ccccc23)ccc1/C═C\c1ccc(/N═N\c2ccc(O)c3ccccc23)cc1S(═O)(═O)O (iKeap59),CC1=NN(c2ccc(S(═O)(═O)O)cc2)C(═O)[C@@H]1/N═N\c1ccc(-c2ccc(/N═N\c3ccc(O)c(C(═O)O)c3)c(C)c2)cc1C(iKeap58),Nc1ccc2cc(S(═O)(═O)O)cc(O)c2c1/N═N\c1ccc(-c2ccc(/N═N\c3c(N)ccc4cc(S(═O)(═O)O)cc(O)c43)cc2)cc1(iKeap10),O═C(O)c1ccc(-c2ccc([C@H]3CC(═O)Oc4ccc5c(c43)O/C(═C\c3cc(F)c(F)c(F)c3)C5=O)o2)cc1(iKeap71), Nc1ccc(/N═N\c2ccc(/C═C\c3ccc(/N═N\c4ccc(N)c5ccccc45)cc3S(═O)(═O)O)c(S(═O)(═O)O)c2)c2ccccc12 (iKeap45),COc1ccc(C(═O)N2CCC[C@H](C(═O)NNC(═O)c3ccc4ccccc4c3)C2)c2ccccc12(iKeap70),O═C(Nc1ccc2[nH]c(-c3cccc(F)c3)nc2c1)[C@@H]1CCCN(C(═O)c2ccc3[nH]ncc3c2)C1(iKeap64),O═C1c2ccccc2/C(═C\NNc2ccc(C(F)(F)F)cc2[N+](═O)[O—])C(═O)N1Cc1ccc2c(c1)OCO2(iKeap43),CC1(C)Cc2oc3c(cc(NS(═O)(═O)c4ccc5c6c(cccc64)C(═O)N5)c4ccccc34)c2C(═O)C1(iKeap65),O═C1c2ccccc2C(═O)N1Cc1cccc(C(═O)N2CCCC[C@H]2c2nc(-c3ccccc3)no2)c1(iKeap44), O═S(═O)(Nc1cccc(-c2ccc3nnc(-c4cccnc4)n3n2)c1)c1ccc2ccccc2c1(iKeap66),CC(═O)N/C(=Cc1ccc(Cc2nc3c([nH]2)C(═O)c2ccccc2C3=O)cc1)c1nc2c([nH]1)C(═O)c1ccccc1C2=O (iKeap48), O═C(NNc1nc2ccccn2n1)c1cc(-c2cccc3ccccc23)nc2ccccc12(iKeap21),O═C(c1ccccc1)C1=C[C@@H]2[C@@H]3C(═O)N(c4cccc5ccccc54)C(═O)[C@@H]3[C@@H](C(═O)c3ccc(Br)cc3)N2C═C1(iKeap15), O═C(c1cc(-c2ccc3c(c2)OCCO3)nc2ccccc21)N1CCC(Cc2ccccc2)CC1(iKeap41), O═C(C1CCN(c2ccc3nnnn3n2)CC1)N1Cc2ccccc2-c2ccccc2C1 (iKeap41),O═c1cc(-c2ccc(O)cc2)oc2c1c(O)cc1c2[C@H](c2ccc3ncccc3c2)CC(═O)O1(iKeap22),C[C@H]1CCc2c(sc3ncnc(N4CCO[C@@H](CN5C(═O)c6ccccc6C5=O)C4)c23)C1(iKeap73),C[C@@H]1Oc2ccc(C(═O)C3CCN(C(═O)[C@H]4C[C@H]4c4cccc5ccccc45)CC3)cc2NC1=O(iKeap74),O═C(Nc1cccc(-c2nnn[nH]2)c1)[C@@H]1C[C@H]2CCCC[C@H]2N1C(═O)c1ccc2ccccc2c1(iKeap8),O═S(═O)(Nc1nc2ccccc2nc1N1CCC[C@@H](c2nc3ccccc3[nH]2)C1)c1ccccc1(iKeap33), O═C(C1Cc2ccccc2C1)N1CCN(C(═O)C2Cc3ccccc3C2)c2ccccc21(iKeap61),O═C1c2ccccc2C(═O)C(N/N═c2/[nH][nH]/c(=N\NC3=C(C1)C(═O)c4ccccc4C3=O)c3ccccc32)=C1C1(iKeap49), CC(═O)N5CCCC4=CC(NC(═O)c3cccc(NC(═O)C2Cc1ccccc1O2)c3)=CCC45(iKeap19),O═C(OCc2cc([N+](═O)O)cc1COCOc12)c6c5CCC/C(═C/c4ccc3OCOc3c4)c5nc7ccccc67(iKeap47), and O═c3[nH]c2ccc(c1ccccc1)cc2c3=NNc6nc(c4ccccn4)nc5CCCc56(iKeap72), or a pharmaceutically acceptable salt, solvate, hydrate,polymorph, co-crystal, tautomer, stereoisomer, isotopically labeledderivative, or prodrug thereof.
 23. The method claim 22, wherein thecompound is selected from the group consisting of: iKeap1, iKeap2,iKeap3, iKeap4, iKeap5, iKeap6, iKeap7, iKeap8, iKeap10, iKeap11,iKeap12, iKeap13, iKeap14, iKeap15, iKeap18, iKeap22, iKeap23, iKeap24,iKeap25, iKeap27, iKeap29, iKeap30, iKeap31, iKeap32, iKeap33, iKeap36,iKeap38, iKeap41, iKeap43, iKeap45, iKeap46, iKeap48, iKeap49, iKeap50,iKeap52, iKeap55, iKeap56, iKeap58, iKeap59, iKeap69, iKeap71, iKeap73,iKeap74, and iKeap75, or a pharmaceutically acceptable salt, solvate,hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopicallylabeled derivative, or prodrug thereof.
 24. A compound having astructure defined by a Simplified Molecular Input Line Entry System(SMILES) selected from the group consisting of:Cc1cc2oc(═O)cc(COC(═O)[C@H]3CCCN(C(═O)c4ccc5[nH]ncc5c4)C3)c2cc1C(C)C(iKeap20),Cc1ccc(C)c(N2C(═O)c3ccc(-c4nc(-c5ccc6[nH]nnc6c5)no4)cc3C2=O)c1(iKeap27), O═C(Cn1nc(-c2ccccc2)ccc1=O)Nc1cccc(Oc2ccc([N+](═O)[O-])cc2)c1(iKeap51),O═C1C[C@H](C(═O)N2CCC[C@H](NC(═O)c3cc4ccccc4o3)C2)c2ccc(F)cc2N1(iKeap26), Cc1nnnn1-c1cccc(NC(═O)c2c3c(nc4ccccc24)/C(═C\c2ccco2)CC3)c1(iKeap46),O═C1N[C@@H](Cc2nc(-c3cccc(Cn4cnc5ccccc54)c3)no2)C(═O)Nc2ccccc21(iKeap60),O═C([C@H]1C[C@H]2CCCC[C@@H]2N1c1ncccn1)N1CC═C(c2c[nH]c3cc(F)ccc23)CC1(iKeap67),COc1ccc(C(═O)N2CCC[C@H](C(═O)NNC(═O)c3ccc4ccccc4c3)C2)c2ccccc12(iKeap70),O═C(Nc1ccc2[nH]c(-c3cccc(F)c3)nc2c1)[C@@H]1CCCN(C(═O)c2ccc3[nH]ncc3c2)C1(iKeap64),O═C1c2ccccc2/C(═C\NNc2ccc(C(F)(F)F)cc2[N+](═O)[O-])C(═O)N1Cc1ccc2c(c1)OCO2(iKeap43),O═C1c2ccccc2C(═O)N1Cc1cccc(C(═O)N2CCCC[C@H]2c2nc(-c3ccccc3)no2)c1(iKeap44), O═C(NNc1nc2ccccn2n1)c1cc(-c2cccc3ccccc23)nc2ccccc12(iKeap21), O═C(C1CCN(c2ccc3nnnn3n2)CC1)N1Cc2ccccc2-c2ccccc2C1 (iKeap76),C[C@H]1CCc2c(sc3ncnc(N4CCO[C@@H](CN5C(═O)c6ccccc6C5=O)C4)c23)C1(iKeap73),C[C@@H]1Oc2ccc(C(═O)C3CCN(C(═O)[C@H]4C[C@H]4c4cccc5ccccc45)CC3)cc2NC1=O(iKeap74),O═C(Nc1cccc(-c2nnn[nH]2)c1)[C@@H]1C[C@H]2CCCC[C@H]2N1C(═O)c1ccc2ccccc2c1(iKeap8),O═S(═O)(Nc1nc2ccccc2nc1N1CCC[C@@H](c2nc3ccccc3[nH]2)C1)c1ccccc1(iKeap33), and O═C(C1Cc2ccccc2C1)N1CCN(C(═O)C2Cc3ccccc3C2)c2ccccc21(iKeap61)Cc1cc2oc(═O)cc(COC(═O)[C@H]3CCCN(C(═O)c4ccc5[nH]ncc5c4)C3)c2cc1C(C)C(iKeap20),Cc1ccc(C)c(N2C(═O)c3ccc(-c4nc(-c5ccc6[nH]nnc6c5)no4)cc3C2=O)c1(iKeap27), O═C(Cn1nc(-c2ccccc2)ccc1=O)Nc1cccc(Oc2ccc([N+](═O)[O-])cc2)c1(iKeap51),O═C1C[C@H](C(═O)N2CCC[C@H](NC(═O)c3cc4ccccc4o3)C2)c2ccc(F)cc2N1(iKeap26), Cc1nnnn1-c1cccc(NC(═O)c2c3c(nc4ccccc24)/C(═C\c2ccco2)CC3)c1(iKeap46),O═C1N[C@@H](Cc2nc(-c3cccc(Cn4cnc5ccccc54)c3)no2)C(═O)Nc2ccccc21(iKeap60),O═C([C@H]1C[C@H]2CCCC[C@@H]2N1c1ncccn1)N1CC═C(c2c[nH]c3cc(F)ccc23)CC1(iKeap67),COc1ccc(C(═O)N2CCC[C@H](C(═O)NNC(═O)c3ccc4ccccc4c3)C2)c2ccccc12(iKeap70),O═C(Nc1ccc2[nH]c(-c3cccc(F)c3)nc2c1)[C@@H]1CCCN(C(═O)c2ccc3[nH]ncc3c2)C1(iKeap64),O═C1c2ccccc2/C(═C\NNc2ccc(C(F)(F)F)cc2[N+](═O)[O-])C(═O)N1Cc1ccc2c(c1)OCO2(iKeap43),O═C1c2ccccc2C(═O)N1Cc1cccc(C(═O)N2CCCC[C@H]2c2nc(-c3ccccc3)no2)c1(iKeap44), O═C(NNc1nc2ccccn2n1)c1cc(-c2cccc3ccccc23)nc2ccccc12(iKeap21), O═C(C1CCN(c2ccc3nnnn3n2)CC1)N1Cc2ccccc2-c2ccccc2C1 (iKeap76),C[C@H]1CCc2c(sc3ncnc(N4CCO[C@@H](CN5C(═O)c6ccccc6C5=O)C4)c23)C1(iKeap73),C[C@@H]1Oc2ccc(C(═O)C3CCN(C(═O)[C@H]4C[C@H]4c4cccc5ccccc45)CC3)cc2NC1=O(iKeap74),O═C(Nc1cccc(-c2nnn[nH]2)c1)[C@@H]1C[C@H]2CCCC[C@H]2N1C(═O)c1ccc2ccccc2c1(iKeap8),O═S(═O)(Nc1nc2ccccc2nc1N1CCC[C@@H](c2nc3ccccc3[nH]2)C1)c1ccccc1(iKeap33), O═C(C1Cc2ccccc2C1)N1CCN(C(═O)C2Cc3ccccc3C2)c2ccccc21(iKeap61), CC(═O)N5CCCC4=CC(NC(═O)c3cccc(NC(═O)C2Cc1ccccc1O2)c3)=CCC45(iKeap19),O═C(OCc2cc([N+](═O)O)cc1COCOc12)c6c5CCC/C(=C/c4ccc3OCOc3c4)c5nc7ccccc67(iKeap47), and O═c3[nH]c2ccc(c1ccccc1)cc2c3=NNc6nc(c4ccccn4)nc5CCCc56(iKeap72), or a pharmaceutically acceptable salt, solvate, hydrate,polymorph, co-crystal, tautomer, stereoisomer, isotopically labeledderivative, or prodrug thereof.
 25. (canceled)
 26. (canceled)